Bicyclic peptidyl inhibitors

ABSTRACT

The present disclosure provides a large combinatorial library of cell-permeable bicyclic peptides. The bicyclic peptides described herein include the first ring consisted of randomized peptide sequences for potential binding to a target of interest while the second ring featured a family of different cell-penetrating motifs, for both cell penetration and target binding. The library was screened against the IκB kinase α/β (IKKα/β)-binding domain of NF-κB essential modulator (NEMO), resulting in the discovery of several cell-permeable bicyclic peptides which inhibited the NEMO-IKKβ interaction, thereby selectively inhibiting canonical NF-κB signaling in mammalian cells and the proliferation of cisplatin-resistant ovarian cancer cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national stage application filed under 35 U.S.C. §371 of PCT/US2018/054345 filed Oct. 4, 2018, which claims the benefit ofU.S. Provisional Application No. 62/568,221, filed on Oct. 4, 2017,which is incorporated by reference herein in its entirety.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

This invention was made with government support under GM110208,GM122459, and GM008512 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing (filename:CYPT_008_01US_SubSeqList_ST25.txt, date recorded: Aug. 4, 2021, filesize ˜52.1 kilobytes).

BACKGROUND

Protein-protein interaction (PPI) is a fundamental aspect of biologicalprocesses, and such interactions mediate numerous disease. However,intracellular PPIs are challenging targets for current drug modalities(i.e., small molecules and biologics) and represent the largest untappedopportunity for therapeutic development.

NF-κB essential modulator (NEMO) is a regulatory protein of thecanonical NF-κB signaling pathway. During canonical NF-κB signaling,receptor activation at the cell surface results in the formation of anactive inhibitor of KB (IκB)-kinase (IKK) complex consisting of IKKα,IKKβ, and NEMO (which is also called IKKγ). Aberrant activation of thecanonical pathway is implicated in many inflammatory and autoimmunediseases, as well as cancer.

The NEMO-IKK complex has been a challenging target for drug discovery.Small molecule inhibitors do not display high enough potency against theNEMO-IKK interaction to be a viable therapeutic strategy. Therefore,there is still need for developing potent peptidyl inhibitors of theNEMO-IKK interaction as a novel class of anti-inflammatory andanticancer drugs.

BRIEF DESCRIPTIONS OF THE FIGURES

FIG. 1 depicts structures of the bicyclic peptide library, hit No. 4,and peptide 7 Amino acid residues in peptide 7 are numbered from N- toC-terminus. The CPP sequence is shown in red, whereas the residuesmodified during optimization are shown in blue color. B, β-alanine; CPP,cell-penetrating peptide; Hmb, hydroxylmethylbenzoyl; Fra,propargylglycine; Δ, L-2,3-diaminopropionic acid (Dap).

FIGS. 2A-2E illustrate the inhibition of the NEMO-IKKα/β interaction andNF-κB signaling by peptide 7 and control peptides. FIG. 2A shows theinhibition of the NEMO-IKKβ interaction as monitored by the HTRF assay.FIG. 2B shows the cellular uptake efficiency of FITC-labeled peptidesinto HeLa cells as determined by flow cytometry. All values are relativeto that of CPP1 (100%). FIG. 2C shows differential effects of pep-tide 7on the basal (open bars) and TNFα-induced NF-κB activation (closed bars)in HEK293(Luc) cells. *, p<0.001 using Student's t-test. FIG. 2Dcompares peptides 1, 7, 20, and 21 for inhibition of TNFα-inducedluciferase activity in HEK293(Luc) cells. FIG. 2E is a western blotshowing the effect of peptide 7 on IκBα and IKKβ levels in HT29 coloncancer cells in the absence and presence of TNFα.

FIGS. 3A-3C show in silico model of the NEMO-peptide 7 complex. FIG. 3Ashows the overall complex between peptide 7 (shown as green sticks) andNEMO (PDBID: 3BRT; shown as van der Waals surface) with residuescritical for the NEMO-IKKβ interaction shaded pink.

FIG. 3B is a close-up of the interaction between the A ring (in green)and NEMO including the insertion of Tyr-4 into a hydrophobic pocket.FIG. 3C shows a zoom-in view of the charge-charge interactions betweenthe three arginine residues of peptide 7 and acidic residues on NEMO.Basic and acidic residues of NEMO are shown in blue and red,respectively.

FIGS. 4A-4D show the anticancer activity of peptide 7. FIG. 4A showslive-cell confocal microscopic image of A2780 ovarian cancer cells after2-hour treatment with 5 μM FITC-peptide 7. I, FITC fluorescence; II,DIC. FIG. 4B shows viability of ovarian cancer cells (A2780 and CP70)and non-cancerous ovarian cells (OSE) in the presence of increasingconcentrations of peptide 7, as determined by the methylene blue assay.FIG. 4C compares peptide 7 and control peptides for their effect on theviability of A2780 cells. FIG. 4D shows the effect of peptide 7 andcontrol peptides on non-cancerous OSE cells. Viability tests in panel(c) and (d) were performed by the MTT assay.

DETAILED DESCRIPTION

Bicyclic Polypeptides

Disclosed herein, in various embodiments, are bicyclic polypeptides. Thebicyclic polypeptides have a first polypeptide sequence which forms afirst ring (referred to herein as the “A ring”), and a secondpolypeptide sequence which forms a second ring (referred to herein asthe “B ring”). In some embodiments, the bicyclic polypeptides disclosedherein penetrate the cell membrane and are capable of inhibitingintracellular protein-protein interactions.

In some embodiments, the bicyclic polypeptides comprise a first sequencewhich is capable of inhibiting a protein-protein interaction (referredto herein as the “Xm” sequence), and a second sequence which is capableof penetrating a cell membrane (referred to herein as the “CPP”sequence). In various embodiments, the Xm sequence may include aminoacids which influence or participate in cellular penetration. Similarly,in embodiments, the CPP sequence may include amino acids whichparticipate in inhibiting a protein-protein interaction.

In various embodiments, the disclosure provides for bicyclicpolypeptides according to Formula 1A or 1B:

wherein:

-   -   CPP is a cell-penetrating peptide sequence;    -   Xm is a peptide sequence that binds to a NF-κB essential        modulator (NEMO) protein;    -   L is a linker moiety; and    -   each of R₁, R₂, and R₃ are, independently, a bonding moiety,    -   wherein the bonding moiety is formed when Xm, CPP, or a        combination thereof, covalently bind to L to form the bicyclic        polypeptide.

In various embodiments, the disclosure provides for bicyclicpolypeptides according to Formula 1C or 1D:

wherein:

-   -   CPP is a cell-penetrating peptide sequence;    -   Xm is a peptide sequence that binds to a NF-κB essential        modulator (NEMO) protein;    -   AA_(L) at each instance is an amino acid;    -   p is selected from a number from 0 to 3 (e.g., 0, 1, 2, or 3);    -   L is a linker moiety; and    -   each of R₁, R₂, and R₃ are independently, a bonding moiety,        wherein the bonding moiety is formed when Xm, CPP, AA_(L) or a        combination thereof, covalently bind to L to form the bicyclic        polypeptide.

Non-limiting examples of bicyclic polypeptides of the present disclosureare provided in Example 1.

Xm Sequence

As discussed above, the bicyclic polypeptides disclosed herein comprisea sequence which is capable of binding to a NEMO protein (“Xm”). Infurther embodiments, the Xm sequence binds the IKKα/β-binding domain onNEMO. In such embodiments, the Xm sequence can be an appropriatecombination of amino acids that binds to IKKα/β-binding domain on NEMO

Suitable amino acid sequences in the Xm sequence for use in the bicyclicpolypeptides and methods described herein can include naturallyoccurring sequences, modified sequences, and synthetic sequences. Inembodiments, the total number of amino acids in the Xm may be in therange of from 3 to about 20 amino acids, e.g., about 4, about 5, about6, about 7, about 8, about 9, about 10, about 11, about 12, about 13,about 14, about 15, about 16, about 17, about 18, and about 19 aminoacids, inclusive of all ranges and subranges therebetween. In someembodiments, the Xm disclosed herein comprise about 4 to about to about13 amino acids. In particular embodiments, the Xm disclosed hereincomprise about 4 to about 10 amino acids, or about 4 to about 8 aminoacids. In other particular embodiments, the Xm disclosed herein compriseabout 6 amino acids.

Each amino acid in the Xm may be a natural or non-natural amino acid.The term “non-natural amino acid” refers to an organic compound that isa congener of a natural amino acid in that it has a structure similar toa natural amino acid so that it mimics the structure and reactivity of anatural amino acid. The non-natural amino acid can be a modified aminoacid, and/or amino acid analog, that is not one of the 20 commonnaturally occurring amino acids or the rare natural amino acidsselenocysteine or pyrrolysine. Non-natural amino acids can also be theD-isomer of the natural amino acids. Examples of suitable amino acidsinclude, but are not limited to, alanine, allosoleucine, arginine,asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine,histidine, isoleucine, leucine, lysine, methionine, napthylalanine,phenylalanine, proline, pyroglutamic acid, serine, threonine,tryptophan, tyrosine, valine, a derivative, or combinations thereof.These, and others, are listed in the Table 1 along with theirabbreviations used herein.

TABLE 1 Amino Acid Abbreviations Abbreviations* Abbreviations* AminoAcid L-amino acid D-amino acid alanine Ala (A) ala (a) allosoleucineAIle aile arginine Arg (R) arg (r) asparagine Asn (N) asn (n) asparticacid Asp (D) asp (d) cysteine Cys (C) cys (c) cyclohexylalanine Cha cha2,3-diaminopropionic acid Dap dap 4-fluorophenylalanine Fpa (Σ) fpaglutamic acid Glu (E) glu (e) glutamine Gln (Q) gln (q) glycine Gly (G)gly (g) histidine His (H) his (h) homoproline Pip (Θ) pip (θ) (akapipecolic acid) isoleucine Ile (I) ile (i) leucine Leu (L) leu (1)lysine Lys (K) lys (k) methionine Met (M) met (m) napthylalanine Nal (Φ)nal (ϕ) norleucine Nle (Ω) nle phenylalanine Phe (F) phe (F)phenylglycine Phg (Ψ) phg 4-(phosphonodifluoro- F₂Pmp (Λ) f₂pmpmethyl)phenylalanine proline Pro (P) pro (p) sarcosine Sar (Ξ) sarselenocysteine Sec (U) sec (u) serine Ser (S) ser (s) threonine Thr (T)thr (y) tyrosine Tyr (Y) tyr (y) tryptophan Trp (W) trp (w) valine Val(V) val (v) *single letter abbreviations: when shown in capital lettersherein it indicates the L-amino acid form, when shown in lower caseherein it indicates the D-amino acid form.

In particular embodiments, the Xm sequence includes one or more aminoacids selected from G, g, W, w, I, i, Y, y, A, and a. In certainembodiments Xm is a 3-7 amino acid sequence comprising at least one W orw. In certain embodiments Xm is a 3-7 amino acid sequence comprising atleast one I or i. In certain embodiments Xm is a 3-7 amino acid sequencecomprising a sequence selected from WI, IW, Wi, iW, wI, Iw, iw, wi. Incertain embodiments Xm is a 3-7 amino acid sequence comprising WI. Infurther embodiments, the Xm is a 4-7 amino acid sequence comprising asequence selected from the group consisting of: GWIY (SEQ ID NO:1);GWIYA (SEQ ID NO:2); GWIYa (SEQ ID NO:50); AGWIY (SEQ ID NO:3); aGWIY(SEQ ID NO:51); AWIYA (SEQ ID NO:4); GAIYA (SEQ ID NO:5); GWAYA (SEQ IDNO:6); GWIAA (SEQ ID NO:7); GWIYA (SEQ ID NO:8); GAIAA (SEQ ID NO:9);and GAAAA (SEQ ID NO:10), and the inverse of such sequences (SEQ ID NOs:52-63).

Cell-Penetrating Peptide Sequence

As discussed above, the bicyclic polypeptides disclosed herein comprisea cell penetrating peptide sequence (“CPP”). The CPP includes any aminosequence which facilitates cellular uptake of the polypeptide conjugatesdisclosed herein.

In some embodiments, the CPPs may include any combination of at leasttwo arginines and at least two hydrophobic amino acids.

In some embodiments, the CPP used in polypeptide conjugates describedherein has a structure comprising Formula 2:(AA _(u))_(m)-AA ₁-AA ₂-AA ₃-AA ₄-(AA _(z))_(n)  2

wherein:

-   -   each of AA₁, AA₂, AA₃, and AA₄, are independently selected from        a D or L amino acid,    -   each of AA_(u) and AA_(z) at each instance and when present, are        independently selected from a D or L amino acid, and    -   m and n are independently selected from a number from 0 to 6;        and

wherein:

-   -   at least two of AA_(u), when present, AA₁, AA₂, AA₃, AA₄, and        AA_(z), when present, are independently arginine, and    -   at least two of AA_(u), when present, AA₁, AA₂, AA₃, AA₄, and        AA_(z), when present, are independently a hydrophobic amino        acid.        (i) In some embodiments, each hydrophobic amino acid is        independently selected from is independently selected from        glycine, alanine, valine, leucine, isoleucine, methionine,        phenylalanine, tryptophan, proline, naphthylalanine,        phenylglycine, homophenylalanine, tyrosine, cyclohexylalanine,        piperidine-2-carboxylic acid, cyclohexylalanine, norleucine,        3-(3-benzothienyl)-alanine, 3-(2-quinolyl)-alanine,        O-benzylserine, 3-(4-(benzyloxy)phenyl)-alanine,        S-(4-methylbenzyl)cysteine, N-(naphthalen-2-yl)glutamine,        3-(1,1′-biphenyl-4-yl)-alanine, tert-leucine, or nicotinoyl        lysine, each of which is optionally substituted with one or more        substituents. The structures of certain of these non-natural        aromatic hydrophobic amino acids (prior to incorporation into        the peptides disclosed herein) are provided below. In particular        embodiments, each hydrophobic amino acid is independently a        hydrophobic aromatic amino acid. In some embodiments, the        aromatic hydrophobic amino acid is naphthylalanine,        3-(3-benzothienyl)-alanine, phenylglycine, homophenylalanine,        phenylalanine, tryptophan, or tyrosine, each of which is        optionally substituted with one or more substituents.

In particular embodiments, the hydrophobic amino acid ispiperidine-2-carboxylic acid, naphthylalanine, tryptophan, orphenylalanine, each of which is optionally substituted with one or moresubstituents.

The optional substituent can be any atom or group which does notsignificantly reduce the cytosolic delivery efficiency of the CPP, e.g.,a substituent that does not reduce relative cytosolic deliveryefficiency to less than that of c(FΦRRRRQ) (SEQ ID NO: 65). In someembodiments, the optional substituent can be a hydrophobic substituentor a hydrophilic substituent. In certain embodiments, the optionalsubstituent is a hydrophobic substituent. In some embodiments, thesubstituent increases the solvent-accessible surface area (as definedherein) of the hydrophobic amino acid. In some embodiments, thesubstituent can be a halogen, alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, heterocyclyl, aryl, heteroaryl, alkoxy,aryloxy, acyl, alkylcarbamoyl, alkylcarboxamidyl, alkoxycarbonyl,alkylthio, or arylthio. In some embodiments, the substituent is ahalogen.

Amino acids having higher hydrophobicity values can be selected toimprove cytosolic delivery efficiency of a CPP relative to amino acidshaving a lower hydrophobicity value. In some embodiments, eachhydrophobic amino acid independently has a hydrophobicity value which isgreater than that of glycine. In other embodiments, each hydrophobicamino acid independently is a hydrophobic amino acid having ahydrophobicity value which is greater than that of alanine. In stillother embodiments, each hydrophobic amino acid independently has ahydrophobicity value which is greater or equal to phenylalanine.Hydrophobicity may be measured using hydrophobicity scales known in theart. Table 2 below lists hydrophobicity values for various amino acidsas reported by Eisenberg and Weiss (Proc. Natl. Acad. Sci. U.S.A 1984;81(1):140-144), Engleman, et al. (Ann. Rev. of Biophys. Biophys. Chem..1986; 1986(15):321-53), Kyte and Doolittle (J. Mol. Biol. 1982;157(1):105-132), Hoop and Woods (Proc. Natl. Acad. Sci. U.S.A. 1981;78(6):3824-3828), and Janin (Nature. 1979; 277(5696):491-492), theentirety of each of which is herein incorporated by reference in itsentirety. In particular embodiments, hydrophobicity is measured usingthe hydrophobicity scale reported in Engleman, et al.

TABLE 2 Eisenberg Kyrie Hoop Amino and Engleman and and Acid Group Weisset al. Doolittle Woods Janin Ile Nonpolar 0.73 3.1 4.5 −1.8 0.7 PheNonpolar 0.61 3.7 2.8 −2.5 0.5 Val Nonpolar 0.54 2.6 4.2 −1.5 0.6 LeuNonpolar 0.53 2.8 3.8 −1.8 0.5 Trp Nonpolar 0.37 1.9 −0.9 −3.4 0.3 MetNonpolar 0.26 3.4 1.9 −1.3 0.4 Ala Nonpolar 0.25 1.6 1.8 −0.5 0.3 GlyNonpolar 0.16 1.0 −0.4 0.0 0.3 Cys Unch/Polar 0.04 2.0 2.5 −1.0 0.9 TyrUnch/Polar 0.02 −0.7 −1.3 −2.3 −0.4 Pro Nonpolar −0.07 −0.2 −1.6 0.0−0.3 Thr Unch/Polar −0.18 1.2 −0.7 −0.4 −0.2 Ser Unch/Polar −0.26 0.6−0.8 0.3 −0.1 His Charged −0.40 −3.0 −3.2 −0.5 −0.1 Glu Charged −0.62−8.2 −3.5 3.0 −0.7 Asn Unch/Polar −0.64 −4.8 −3.5 0.2 −0.5 GlnUnch/Polar −0.69 −4.1 −3.5 0.2 −0.7 Asp Charged −0.72 −9.2 −3.5 3.0 −0.6Lys Charged −1.10 −8.8 −3.9 3.0 −1.8 Arg Charged −1.80 −12.3 −4.5 3.0−1.4

The chirality of the amino acids can be selected to improve cytosolicuptake efficiency. In some embodiments, at least two of the amino acidshave the opposite chirality. In some embodiments, the at least two aminoacids having the opposite chirality can be adjacent to each other. Insome embodiments, at least three amino acids have alternatingstereochemistry relative to each other. In some embodiments, the atleast three amino acids having the alternating chirality relative toeach other can be adjacent to each other. In some embodiments, at leasttwo of the amino acids have the same chirality. In some embodiments, theat least two amino acids having the same chirality can be adjacent toeach other. In some embodiments, at least two amino acids have the samechirality and at least two amino acids have the opposite chirality. Insome embodiments, the at least two amino acids having the oppositechirality can be adjacent to the at least two amino acids having thesame chirality. Accordingly, in some embodiments, adjacent amino acidsin the CPP can have any of the following sequences: D-L; L-D; D-L-L-D;L-D-D-L; L-D-L-L-D; D-L-D-D-L; D-L-L-D-L; or L-D-D-L-D.

In some embodiments, an arginine is adjacent to a hydrophobic aminoacid. In some embodiments, the arginine has the same chirality as thehydrophobic amino acid. In some embodiments, at least two arginines areadjacent to each other. In still other embodiments, three arginines areadjacent to each other. In some embodiments, at least two hydrophobicamino acids are adjacent to each other. In other embodiments, at leastthree hydrophobic amino acids are adjacent to each other. In otherembodiments, the CPPs described herein comprise at least two consecutivehydrophobic amino acids and at least two consecutive arginines. Infurther embodiments, one hydrophobic amino acid is adjacent to one ofthe arginines. In still other embodiments, the CPPs described hereincomprise at least three consecutive hydrophobic amino acids and thereconsecutive arginines. In further embodiments, one hydrophobic aminoacid is adjacent to one of the arginines. These various combinations ofamino acids can have any arrangement of D and L amino acids, e.g., thesequences described above.

In some embodiments, any four adjacent amino acids in the CPPs describedherein (e.g., the CPPs according to Formula 2) can have one of thefollowing sequences: AA_(H2)-AA_(H1)-R-r, AA_(H2)-AA_(H1)-r-R,R-r-AA_(H1)-AA_(H2), or r-R-AA_(H1)-AA_(H2), wherein each of AA_(H1) andAA_(H2) are independently a hydrophobic amino acid. Accordingly, in someembodiments, the CPPs used in the polypeptide conjugates describedherein have a structure according any of Formula 3A-D:

wherein:

each of AA_(H1) and AA_(H2) are independently a hydrophobic amino acid;

at each instance and when present, each of AA_(U) and AA_(Z) areIndependently any Amino acid; and

m and n are independently selected from a number from 0 to 6.

In some embodiments, the total number of amino acids (including r, R,AA_(H1), AA_(H2)), in the CPPs of Formula 3-A to 3-D are in the range of4 to 10, e.g., 6. In some embodiments, the total number of amino acidsis 4. In some embodiments, the total number of amino acids is 5. In someembodiments, the total number of amino acids is 6. In some embodiments,the total number of amino acids is 7. In some embodiments, the totalnumber of amino acids is 8. In some embodiments, the total number ofamino acids is 9. In some embodiments, the total number of amino acidsis 10.

In some embodiments, the sum of m and n is from 2 to 6. In someembodiments, the sum of m and n is 2. In some embodiments, the sum of mand n is 3. In some embodiments, the sum of m and n is 4. In someembodiments, the sum of m and n is 5. In some embodiments, the sum of mand n is 6. In some embodiments, m is 0. In some embodiments, m is 1. Insome embodiments, m is 2. In some embodiments, m is 3. In someembodiments, m is 4. In some embodiments, m is 5. In some embodiments, mis 6. In some embodiments, n is 0. In some embodiments, n is 1. In someembodiments, n is 2. In some embodiments, n is 3. In some embodiments, nis 4. In some embodiments, n is 5. In some embodiments, n is 6.

In some embodiments, each hydrophobic amino acid is independentlyselected from glycine, alanine, valine, leucine, isoleucine, methionine,phenylalanine, tryptophan, proline, naphthylalanine, phenylglycine,homophenylalanine, tyrosine, cyclohexylalanine, piperidine-2-carboxylicacid, or norleucine, each of which is optionally substituted with one ormore substituents. In particular embodiments, each hydrophobic aminoacid is independently a hydrophobic aromatic amino acid. In someembodiments, the aromatic hydrophobic amino acid ispiperidine-2-carboxylic acid, naphthylalanine, phenylglycine,homophenylalanine, phenylalanine, tryptophan, or tyrosine, each of whichis optionally substituted with one or more substituents. In particularembodiments, the hydrophobic amino acid is piperidine-2-carboxylic acid,naphthylalanine, tryptophan, or phenylalanine, each of which isoptionally substituted with one or more substituents.

In some embodiments, each of AA_(H1) and AA_(H2) are independently ahydrophobic amino acid having a hydrophobicity value which is greaterthan that of glycine. In other embodiments, each of AA_(H1) and AA_(H2)are independently a hydrophobic amino acid having a hydrophobicity valuewhich is greater than that of alanine. In still other embodiments, eachof AA_(H1) and AA_(H2) are independently an hydrophobic amino acidhaving a hydrophobicity value which is greater than that ofphenylalanine, e.g., as measured using the hydrophobicity scalesdescribed above, including Eisenberg and Weiss (Proc. Natl. Acad. Sci.U.S.A 1984; 81(1):140-144), Engleman, et al. (Ann. Rev. of Biophys.Biophys. Chem.. 1986; 1986(15):321-53), Kyte and Doolittle (J. Mol.Biol. 1982; 157(1):105-132), Hoop and Woods (Proc. Natl. Acad. Sci.U.S.A. 1981; 78(6):3824-3828), and Janin (Nature. 1979;277(5696):491-492), (see Table 1 above). In particular embodiments,hydrophobicity is measured using the hydrophobicity scale reported inEngleman, et al.

The presence of a hydrophobic amino acid on the N- or C-terminal of aD-Arg or L-Arg, or a combination thereof, has also found to improve thecytosolic uptake of the CPP (and the attached cargo). For example, insome embodiments, the CPPs disclosed herein may include AA_(H1)-D-Arg orD-Arg-AA_(H1). In other embodiments, the CPPs disclosed herein mayinclude AA_(H1)-L-Arg or L-Arg-AA_(H1). In some embodiments, thepresence of the hydrophobic amino acid on the N- or C-terminal of theD-Arg or L-Arg, or a combination thereof, in the CPP improves thecytosolic delivery efficiency by about 1.1 fold to about 30 fold,compared to an otherwise identical sequence, e.g., about 1.2, about 1.3,about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0,about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about8.5, about 9.0, about 10, about 10.5, about 11.0, about 11.5, about12.0, about 12.5, about 13.0, about 13.5, about 14.0, about 14.5, about15.0, about 15.5, about 16.0, about 16.5, about 17.0, about 17.5, about18.0, about 18.5, about 19.0, about 19.5, about 20, about 20.5, about21.0, about 21.5, about 22.0, about 22.5, about 23.0, about 23.5, about24.0, about 24.5, about 25.0, about 25.5, about 26.0, about 26.5, about27.0, about 27.5, about 28.0, about 28.5, about 29.0, or about 29.5fold, inclusive of all values and subranges therebetween. In someembodiments, the presence of the hydrophobic amino acid on the N- and/orC-terminal of the D-Arg and/or L-Arg in the CPP improves the cytosolicuptake efficiency by about 20 fold.

The size of the hydrophobic amino acid on the N- or C-terminal of theD-Arg or an L-Arg, or a combination thereof (i.e., AA_(H1)), may beselected to improve cytosolic delivery efficiency of the CPP. Forexample, a larger hydrophobic amino acid on the N- or C-terminal of aD-Arg or L-Arg, or a combination thereof, improves cytosolic deliveryefficiency compared to an otherwise identical sequence having a smallerhydrophobic amino acid. The size of the hydrophobic amino acid can bemeasured in terms of molecular weight of the hydrophobic amino acid, thesteric effects of the hydrophobic amino acid, the solvent-accessiblesurface area (SASA) of the side chain, or combinations thereof. In someembodiments, the size of the hydrophobic amino acid is measured in termsof the molecular weight of the hydrophobic amino acid, and the largerhydrophobic amino acid has a side chain with a molecular weight of atleast about 90 g/mol, or at least about 130 g/mol, or at least about 141g/mol. In other embodiments, the size of the amino acid is measured interms of the SASA of the hydrophobic side chain, and the largerhydrophobic amino acid has a side chain with a SASA greater thanalanine, or greater than glycine. In other embodiments, AA_(H1) has ahydrophobic side chain with a SASA greater than or equal to aboutpiperidine-2-carboxylic acid, greater than or equal to about tryptophan,greater than or equal to about phenylalanine, or equal to or greaterthan about naphthylalanine. In some embodiments, AA_(H1) has a sidechain side with a SASA of at least about 200 Å², at least about 210 Å2,at least about 220 Å², at least about 240 Å², at least about 250 Å², atleast about 260 Å², at least about 270 Å², at least about 280 Å², atleast about 290 Å², at least about 300 Å², at least about 310 Å², atleast about 320 Å², or at least about 330 Å². In some embodiments, AAH₂has a side chain side with a SASA of at least about 200 Å², at leastabout 210 Å2, at least about 220 Å², at least about 240 Å², at leastabout 250 Å², at least about 260 Å², at least about 270 Å², at leastabout 280 Å², at least about 290 Å², at least about 300 Å², at leastabout 310 Å², at least about 320 Å², or at least about 330 Å². In someembodiments, the side chains of AAH₁ and AAH₂ have a combined SASA of atleast about 350 Å², at least about 360 Å², at least about 370 Å², atleast about 380 Å2, at least about 390 Å², at least about 400 Å², atleast about 410 Å², at least about 420 Å², at least about 430 Å², atleast about 440 Å², at least about 450 Å², at least about 460 Å², atleast about 470 Å², at least about 480 Å², at least about 490 Å²,greater than about 500 Å², at least about 510 Å², at least about 520 Å²,at least about 530 Å², at least about 540 Å², at least about 550 Å², atleast about 560 Å², at least about 570 Å², at least about 580 Å², atleast about 590 Å², at least about 600 Å², at least about 610 Å², atleast about 620 Å², at least about 630 Å², at least about 640 Å²,greater than about 650 Å², at least about 660 Å², at least about 670 Å²,at least about 680 Å², at least about 690 Å², or at least about 700 Å².In some embodiments, AA_(H2) is a hydrophobic amino acid with a sidechain having a SASA that is less than or equal to the SASA of thehydrophobic side chain of AA_(H1). By way of example, and not bylimitation, a CPP having a Nal-Arg motif exhibits improved cytosolicdelivery efficiency compared to an otherwise identical CPP having aPhe-Arg motif; a CPP having a Phe-Nal-Arg motif exhibits improvedcytosolic delivery efficiency compared to an otherwise identical CPPhaving a Nal-Phe-Arg motif; and a phe-Nal-Arg motif exhibits improvedcytosolic delivery efficiency compared to an otherwise identical CPPhaving a nal-Phe-Arg motif.

As used herein, “hydrophobic surface area” or “SASA” refers to thesurface area (reported as square Ångstroms; Å²) of an amino acid sidechain that is accessible to a solvent. In particular embodiments, SASAis calculated using the ‘rolling ball’ algorithm developed by Shrake &Rupley (J Mol Biol. 79 (2): 351-71), which is herein incorporated byreference in its entirety for all purposes. This algorithm uses a“sphere” of solvent of a particular radius to probe the surface of themolecule. A typical value of the sphere is 1.4 Å, which approximates tothe radius of a water molecule.

SASA values for certain side chains are shown below in Table 3. Incertain embodiments, the SASA values described herein are based on thetheoretical values listed in Table 3 below, as reported by Tien, et al.(PLOS ONE 8(11): e80635. https://doi.org/10.1371/journal.pone.0080635,which is herein incorporated by reference in its entirety for allpurposes.

TABLE 3 Miller et Rose et Residue Theoretical Empirical al. (1987) al.(1985) Alanine 129.0 121.0 113.0 118.1 Arginine 274.0 265.0 241.0 256.0Asparagine 195.0 187.0 158.0 165.5 Aspartate 193.0 187.0 151.0 158.7Cysteine 167.0 148.0 140.0 146.1 Glutamate 223.0 214.0 183.0 186.2Glutamine 225.0 214.0 189.0 193.2 Glycine 104.0 97.0 85.0 88.1 Histidine224.0 216.0 194.0 202.5 Isoleucine 197.0 195.0 182.0 181.0 Leucine 201.0191.0 180.0 193.1 Lysine 236.0 230.0 211.0 225.8 Methionine 224.0 203.0204.0 203.4 Phenylalanine 240.0 228.0 218.0 222.8 Proline 159.0 154.0143.0 146.8 Serine 155.0 143.0 122.0 129.8 Threonine 172.0 163.0 146.0152.5 Tryptophan 285.0 264.0 259.0 266.3 Tyrosine 263.0 255.0 229.0236.8 Valine 174.0 165.0 160.0 164.5

In some embodiments, the CPP does not include a hydrophobic amino acidon the N- and/or C-terminal of AA_(H2)-AA_(H1)-R-r, AA_(H2)-AA_(H1)-r-R,R-r-AA_(H1)-AA_(H2), or r-R-AA_(H1)-AA_(H2). In alternative embodiments,the CPP does not include a hydrophobic amino acid having a side chainwhich is larger (as described herein) than at least one of AA_(H1) orAA_(H2). In further embodiments, the CPP does not include a hydrophobicamino acid with a side chain having a surface area greater than AA_(H1).For example, in embodiments in which at least one of AA_(H1) or AA_(H2)is phenylalanine, the CPP does not further include a naphthylalanine(although the CPP include at least one hydrophobic amino acid which issmaller than AA_(H1) and AA_(H2), e.g., leucine). In still otherembodiments, the CPP does not include a naphthylalanine in addition tothe hydrophobic amino acids in AA_(H2)-AA_(H1)-R-r, AA_(H2)-AA_(H1)-r-R,R-r-AA_(H1)-AA_(H2), or r-R-AA_(H1)-AA_(H2).

The chirality of the amino acids (i.e., D or L amino acids) can beselected to improve cytosolic delivery efficiency of the CPP (and theattached cargo as described below). In some embodiments, the hydrophobicamino acid on the N- or C-terminal of an arginine (e.g., AA_(H1)) hasthe same or opposite chirality as the adjacent arginine. In someembodiments, AA_(H1) has the opposite chirality as the adjacentarginine. For example, when the arginine is D-arg (i.e. “r”), AA_(H1) isa D-AA_(H1), and when the arginine is L-Arg (i.e., “R”), AA_(H1) is aL-AA_(H1). Accordingly, in some embodiments, the CPPs disclosed hereinmay include at least one of the following motifs: D-AA_(H1)-D-arg,D-arg-D-AA_(H1), L-AA_(H1)-L-Arg, or L-Arg-LAA_(H1). In particularembodiments, when arginine is D-arg, AA_(H) can be D-nal, D-trp, orD-phe. In another non-limiting example, when arginine is L-Arg, AA_(H)can be L-Nal, L-Trp, or L-Phe.

In some embodiments, the CPPs described herein include three arginines.Accordingly, in some embodiments, the CPPs described herein include oneof the following sequences: AA_(H2)-AA_(H1)-R-r-R,AA_(H2)-AA_(H1)-R-r-r, AA_(H2)-AA_(H1)-r-R-R, AA_(H2)-AA_(H1)-r-R-r,R-R-r-AA_(H1)-AA_(H2), r-R-r-AA_(H1)-AA_(H2), r-r-R-AA_(H1)-AA_(H2), or,R-r-R-AA_(H1)-AA_(H2). In particular embodiments, the CPPs have one ofthe following sequences AA_(H2)-AA_(H1)-R-r-R, AA_(H2)-AA_(H1)-r-R-r,r-R-r-AA_(H1)-AA_(H2), or R-r-R-AA_(H1)-AA_(H2). In some embodiments,the chirality of AAH₁ and AAH₂ can be selected to improve cytosolicuptake efficiency, e.g., as described above, where AAH₁ has the samechirality as the adjacent arginine, and AAH₁ and AAH₂ have the oppositechirality.

In some embodiments, the CPPs described herein include three hydrophobicamino acids. Accordingly, in some embodiments, the CPPs described hereininclude one of the following sequences: AA_(H3)-AA_(H2)-AA_(H1)-R-r,AA_(H3)-AA_(H2)-AA_(H1)-R-r, AA_(H3)-AA_(H2)-AA_(H1)-r-R,AA_(H3)-AA_(H2)-AA_(H1)-r-R, R-r-AA_(H1)-AA_(H2)-AA_(H3),R-r-AA_(H1)-AA_(H2)-AA_(H3), r-R-AA_(H1)-AA_(H2)-AA_(H3), or,r-R-AA_(H1)-AA_(H2)-AA_(H3), wherein AA_(H3) is any hydrophobic aminoacid described above, e.g., piperidine-2-carboxylic acid,naphthylalanine, tryptophan, or phenylalanine. In some embodiments, thechirality of AA_(H1), AA_(H2), and AA_(H3) can be selected to improvecytosolic uptake efficiency, e.g., as described above, where AA_(H1) hasthe same chirality as the adjacent arginine, and AA_(H1) and AA_(H2)have the opposite chirality. In other embodiments, the size of AA_(H1),AA_(H2), and AA_(H3) can be selected to improve cytosolic uptakeefficiency, e.g., as described above, where AA_(H3) has a SAS of lessthan or equal to AA_(H1) and/or AA_(H2).

In some embodiments, AA_(H1) and AA_(H2) have the same or oppositechirality. In certain embodiments, AA_(H1) and AA_(H2) have the oppositechirality. Accordingly, in some embodiments, the CPPs disclosed hereininclude at least one of the following sequences:D-AA_(H2)-L-AA_(H1)-R-r; L-AA_(H2)-D-AA_(H1)-r-R;R-r-D-AA_(H1)-L-AA_(H2); or r-R-L-AA_(H1)-D-AA_(H1), wherein each ofD-AA_(H1) and D-AA_(H2) is a hydrophobic amino acid having a Dconfiguration, and each of L-AA_(H1) and L-AA_(H2) is a hydrophobicamino acid having an L configuration. In some embodiments, each ofD-AA_(H1) and D-AA_(H2) is independently selected from the groupconsisting of D-pip, D-nal, D-trp, and D-phe. In particular embodiments,D-AA_(H1) or D-AA_(H2) is D-nal. In other particular embodiments,D-AA_(H1) is D-nal. In some embodiments, each of L-AA_(H1) and L-AA_(H2)is independently selected from the group consisting of L-Pip, L-Nal,L-Trp, and L-Phe. In particular embodiments, each of L-AA_(H1) andL-AA_(H2) is L-Nal. In other particular embodiments, L-AA_(H1) is L-Nal.

As discussed above, the disclosure provides for various modifications toa cyclic peptide sequence which may improve cytosolic deliveryefficiency. In some embodiments, improved cytosolic uptake efficiencycan be measured by comparing the cytosolic delivery efficiency of theCPP having the modified sequence to a proper control sequence. In someembodiments, the control sequence does not include a particularmodification (e.g., matching chirality of R and AA_(H1)) but isotherwise identical to the modified sequence. In other embodiments, thecontrol has the following sequence: cyclic(FΦRRRRQ) (SEQ ID NO: 65).

As used herein cytosolic delivery efficiency refers to the ability of aCPP to traverse a cell membrane and enter the cytosol. In embodiments,cytosolic delivery efficiency of the CPP is not dependent on a receptoror a cell type. Cytosolic delivery efficiency can refer to absolutecytosolic delivery efficiency or relative cytosolic delivery efficiency.

Absolute cytosolic delivery efficiency is the ratio of cytosolicconcentration of a CPP (or a CPP-cargo conjugate) over the concentrationof the CPP (or the CPP-cargo conjugate) in the growth medium. Relativecytosolic delivery efficiency refers to the concentration of a CPP inthe cytosol compared to the concentration of a control CPP in thecytosol. Quantification can be achieved by fluorescently labeling theCPP (e.g., with a FTIC dye) and measuring the fluorescence intensityusing techniques well-known in the art.

In particular embodiments, relative cytosolic delivery efficiency isdetermined by comparing (i) the amount of a CPP of the inventioninternalized by a cell type (e.g., HeLa cells) to (ii) the amount of thecontrol CPP internalized by the same cell type. To measure relativecytosolic delivery efficiency, the cell type may be incubated in thepresence of a cell-penetrating peptide of the invention for a specifiedperiod of time (e.g., 30 minutes, 1 hour, 2 hours, etc.) after which theamount of the CPP internalized by the cell is quantified using methodsknown in the art, e.g., fluorescence microscopy. Separately, the sameconcentration of the control CPP is incubated in the presence of thecell type over the same period of time, and the amount of the controlCPP internalized by the cell is quantified.

In other embodiments, relative cytosolic delivery efficiency can bedetermined by measuring the IC₅₀ of a CPP having a modified sequence foran intracellular target, and comparing the IC₅₀ of the CPP having themodified sequence to a proper control sequence (as described herein).

Non-limiting examples of suitable cyclic cell penetrating peptide areprovided in Table 4. The CPP sequence in the polypeptides of the presentdisclosure can include any of the CPP sequences provided in Table 4, ora subset of amino acids in the CPPs provided in Table 4.

TABLE 4 ID CPP Sequence PCT 1 FΦRRR (SEQ ID NO: 66) PCT 2FΦRRRC (SEQ ID NO: 67) PCT 3 FΦRRRU (SEQ ID NO: 68) PCT 4RRRΦF (SEQ ID NO: 69) PCT 5 RRRRΦF (SEQ ID NO: 70) PCT 6FΦRRRR (SEQ ID NO: 71) PCT 7 FϕrRrR (SEQ ID NO: 72) PCT 8FϕrRrR (SEQ ID NO: 72) PCT 9 FΦRRRR (SEQ ID NO: 71) PCT 10fΦRrRr (SEQ ID NO: 73) PCT 11 RRFRΦR (SEQ ID NO: 74) PCT 12FRRRRΦ (SEQ ID NO: 75) PCT 13 rRFRΦR (SEQ ID NO: 76) PCT 14RRΦFRR (SEQ ID NO: 77) PCT 15 CRRRRFW (SEQ ID NO: 11) PCT 16FfΦRrRr (SEQ ID NO: 78) PCT 17 FFΦRRRR (SEQ ID NO: 79) PCT 18RFRFRΦR (SEQ ID NO: 80) PCT 19 URRRRFW (SEQ ID NO: 12) PCT 20CRRRRFW (SEQ ID NO: 13) PCT 21 FΦRRRRQK (SEQ ID NO: 81) PCT 22FΦRRRRQC (SEQ ID NO: 82) PCT 23 fΦRrRrR (SEQ ID NO: 83) PCT 24FΦRRRRR (SEQ ID NO: 84) PCT 25 RRRRΦFDΩC (SEQ ID NO: 85) PCT 26FΦRRR (SEQ ID NO: 66) PCT 27 FWRRR (SEQ ID NO: 14) PCT 28RRRΦF (SEQ ID NO: 69) PCT 29 RRRWF (SEQ ID NO: 15) SAR 1FΦRRRR (SEQ ID NO: 71) SAR 19 FFRRR (SEQ ID NO: 16) SAR 20FFrRr (SEQ ID NO: 86) SAR 21 FFRrR (SEQ ID NO: 87) SAR 22FRFRR (SEQ ID NO: 17) SAR 23 FRRFR (SEQ ID NO: 18) SAR 24FRRRF (SEQ ID NO: 19) SAR 25 GΦRRR (SEQ ID NO: 88) SAR 26FFFRA (SEQ ID NO: 20) SAR 27 FFFRR (SEQ ID NO: 21) SAR 28FFRRRR (SEQ ID NO: 22) SAR 29 FRRFRR (SEQ ID NO: 23) SAR 30FRRRFR (SEQ ID NO: 24) SAR 31 RFFRRR (SEQ ID NO: 25) SAR 32RFRRFR (SEQ ID NO: 26) SAR 33 FRFRRR (SEQ ID NO: 27) SAR 34FFFRRR (SEQ ID NO: 28) SAR 35 FFRRRF (SEQ ID NO: 29) SAR 36FRFFRR (SEQ ID NO: 30) SAR 37 RRFFFR (SEQ ID NO: 31) SAR 38FFRFRR (SEQ ID NO: 32) SAR 39 FFRRFR (SEQ ID NO: 33) SAR 40FRRFFR (SEQ ID NO: 34) SAR 41 FRRFRF (SEQ ID NO: 35) SAR 42FRFRFR (SEQ ID NO: 36) SAR 43 RFFRFR (SEQ ID NO: 37) SAR 44GΦRRRR (SEQ ID NO: 89) SAR 45 FFFRRRR (SEQ ID NO: 38) SAR 46RFFRRRR (SEQ ID NO: 39) SAR 47 RRFFRRR (SEQ ID NO: 40) SAR 48RFFFRRR (SEQ ID NO: 41) SAR 49 RRFFFRR (SEQ ID NO: 42) SAR 50FFRRFRR (SEQ ID NO: 43) SAR 51 FFRRRRF (SEQ ID NO: 44) SAR 52FRRFFRR (SEQ ID NO: 45) SAR 53 FFFRRRRR (SEQ ID NO: 46) SAR 54FFFRRRRRR (SEQ ID NO: 47) SAR 55 FΦRrRr (SEQ ID NO: 90) SAR 56XXRRRR (SEQ ID NO: 48) SAR 57 FfFRrR (SEQ ID NO: 91) SAR 58fFfrRr (SEQ ID NO: 92) SAR 59 fFfRrR (SEQ ID NO: 93) SAR 60FfFrRr (SEQ ID NO: 94) SAR 61 fFϕnRr (SEQ ID NO: 95) SAR 62KΦfrRr (SEQ ID NO: 96) SAR 63 ϕKfrRr (SEQ ID NO: 97) SAR 64FΦrRr (SEQ ID NO: 98) SAR 65 fΦrRr (SEQ ID NO: 99) SAR 66Ac-(Lys-fFRrRrD) (SEQ ID NO: 100) SAR 67Ac-(Dap-fFRrRrD) (SEQ ID NO: 101) SAR 68

 (SEQ ID NO: 102) SAR 69

 (SEQ ID NO: 103) SAR 70

 (SEQ ID NO: 104) SAR 71

 (SEQ ID NO: 105) Pin1 15 Pip-Nal-Arg-Glu-arg-arg-glu (SEQ ID NO: 106)Pin1 16 Pip-Nal-Arg-Arg-arg-arg-glu (SEQ ID NO: 107) Pin1 17Pip-Nal-Nal-Arg-arg-arg-glu (SEQ ID NO: 108) Pin1 18Pip-Nal-Nal-Arg-arg-arg-Glu (SEQ ID NO: 109) Pin1 19Pip-Nal-Phe-Arg-arg-arg-glu (SEQ ID NO: 110) Pin1 20Pip-Nal-Phe-Arg-arg-arg-Glu (SEQ ID NO: 111) Pin1 21Pip-Nal-phe-Arg-arg-arg-glu) (SEQ ID NO: 112) Pin1 22Pip-Nal-phe-Arg-arg-arg-Glu (SEQ ID NO: 113) Pin1 23Pip-Nal-nal-Arg-arg-arg-Glu (SEQ ID NO: 114) Pin1 24Pip-Nal-nal-Arg-arg-arg-glu (SEQ ID NO: 115) Rev-13[Pim-RQRR-Nlys]GRRR^(b) (SEQ ID NO: 116) hLF

 (SEQ ID NO: 117) cTat [KrRrGrKkrE]^(c) (SEQ ID NO: 118) cR10[KrRrRrRrRrRE]^(c) (SEQ ID NO: 119) L-50[RVRTRGKRRIRRpP] (SEQ ID NO: 120) L-51 [RTRTRGKRRIRVpP] (SEQ ID NO: 121)[WR]₄ [WRWRWRWR] (SEQ ID NO: 49) MCoTI-II

 (SEQ ID NO: 122) Rotstein et al.[P-Cha-r-Cha-r-Cha-r-Cha-r-G]^(d) (SEQ ID NO: 123) Chem. Eur. J. 2011Lian et al. J. Tm(SvP-F₂Pmp-H)-Dap-(FΦRRRR-Dap)^(f) (SEQ ID NO: 124)Am. Chem. Soc. 2014 Lian et al. J. [Tm(a-Sar-D-pThr-Pip-ΦRAa)-Dap-Am. Chem. Soc. (FΦRRRR-Dap)]^(f) (SEQ ID NO: 125) 2014 IA8b[CRRSRRGCGRRSRRCG]^(g) (SEQ ID NO: 127) Dod-[R₅][K(Dod)RRRR] (SEQ ID NO: 128) LK-3                 (SEQ ID NO: 128)

(SEQ ID NO: 129) RRRR-[KRRRE]^(c) (SEQ ID NO: 130)RRR-[KRRRRE]^(c) (SEQ ID NO: 131) RR-[KRRRRRE]^(c) (SEQ ID NO: 132)R-[KRRRRRRE]^(c) (SEQ ID NO: 133) [CR]₄ [CRCRCRCR] (SEQ ID NO: 134) cyc3[Pra-LRKRLRKFRN-AzK]^(h) (SEQ ID NO: 135) PMBT-Dap-[Dap-Dap-f-L-Dap-Dap-T] (SEQ ID NO: 136) GPMBT-Agp-[Dap-Agp-f-L-Agp-Agp-T] (SEQ ID NO: 137) CPP1FΦRRRR (SEQ ID NO: 71) CPP12 FfΦRrRr (SEQ ID NO: 78) CPP9fΦRrRr (SEQ ID NO: 73) CPP11 fΦRrRrR (SEQ ID NO: 83) CPP18FϕrRrR (SEQ ID NO: 72) CPP13 FϕrRrR (SEQ ID NO: 72) CPP6FΦRRRRR (SEQ ID NO: 84) CPP3 RRFRΦR (SEQ ID NO: 74) CPP7FFΦRRRR (SEQ ID NO: 79) CPP8 RFRFRΦR (SEQ ID NO: 80) CPP5FΦRRR (SEQ ID NO: 66) CPP4 FRRRRΦ (SEQ ID NO: 75) CPP10rRFRΦR (SEQ ID NO: 76) CPP2 RRΦFRR (SEQ ID NO: 77) Φ,L-2-naphthylalanine; Pim, pimelic acid; Nlys, lysine peptoid residue;D-pThr, D-phosphothreonine; Pip, L-piperidine-2-carboxylic acid; Cha,L-3-cyclohexyl-alanine; Tm, trimesic acid; Dap, L-2,3-diaminopropionicacid; Sar, sarcosine; F₂Pmp, L-difluorophosphonomethyl phenylalanine;Dod, dodecanoyl; Pra, L-propargylglycine; AzK,L-6-Azido-2-amino-hexanoic; Agp, L-2-amino-3-guanidinylpropionic acid;^(b)Cyclization between Pim and Nlys; ^(c)Cyclization between Lys andGlu; ^(d)Macrocyclization by multicomponent reaction with aziridinealdehyde and isocyanide; ^(e)Cyclization between the main-chain of Glnresidue; ^(f)N-terminal amine and side chains of two Dap residuesbicyclized with Tm; ^(g)Three Cys side chains bicyclized withtris(bromomethyl)benzene; ^(h)Cyclization by the click reaction betweenPra and Azk.Φ, L-2-naphthylalanine; Pim, pimelic acid; Nlys, lysine peptoid residue;D-pThr, D-phosphothreonine; Pip, L-piperidine-2-carboxylic acid; Cha,L-3-cyclohexyl-alanine; Tm, trimesic acid; Dap, L-2,3-diaminopropionicacid; Sar, sarcosine; F₂Pmp, L-difluorophosphonomethyl phenylalanine;Dod, dodecanoyl; Pra, L-propargylglycine; AzK,L-6-Azido-2-amino-hexanoic; Agp, L-2-amino-3-guanidinylpropionic acid;^(b)Cyclization between Pim and Nlys; ^(c)Cyclization between Lys andGlu; ^(d)Macrocyclization by multicomponent reaction with aziridinealdehyde and isocyanide; ^(e)Cyclization between the main-chain of Glnresidue; ^(f)N-terminal amine and side chains of two Dap residuesbicyclized with Tm; ^(g)Three Cys side chains bicyclized withtris(bromomethyl)benzene; ^(h)Cyclization by the click reaction betweenPra and Azk.

The cell-penetrating peptide sequences of the present disclose caninclude any of those disclosed in US 2017/0355730 Å1, WO/2018/098231(and the US patent application publication related thereto), and U.S.Provisional application No. 62/669,146 (and the US patent applicationpublication related thereto), each of which are herein incorporated byreference in its entirety for all purposes.

In certain embodiments, the cytosolic delivery efficiency of aparticular CPP used in the bicyclic polypeptide of the claimedinvention, may also be dependent on the sequence of Xm. In furtherembodiments of the invention, a particular CPP/Xm bicyclic polypeptidemay have an improved cytosolic delivery efficiency of about 1.1 fold toabout 30 fold, compared to an bicyclic polypeptide having an identicalCPP and a different Xm, e.g., about 1.2, about 1.3, about 1.4, about1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.5,about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, about 9.0,about 10, about 10.5, about 11.0, about 11.5, about 12.0, about 12.5,about 13.0, about 13.5, about 14.0, about 14.5, about 15.0, about 15.5,about 16.0, about 16.5, about 17.0, about 17.5, about 18.0, about 18.5,about 19.0, about 19.5, about 20, about 20.5, about 21.0, about 21.5,about 22.0, about 22.5, about 23.0, about 23.5, about 24.0, about 24.5,about 25.0, about 25.5, about 26.0, about 26.5, about 27.0, about 27.5,about 28.0, about 28.5, about 29.0, or about 29.5 fold inclusive of allvalues and subranges therebetween. In further embodiments of theinvention, a particular CPP/Xm bicyclic polypeptide may have an improvedcytosolic delivery efficiency of about 1.1 fold to about 30 fold,compared to an bicyclic polypeptide having an identical Xm and adifferent CPP, e.g., about 1.2, about 1.3, about 1.4, about 1.5, about1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.5, about 3.0,about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about6.5, about 7.0, about 7.5, about 8.0, about 8.5, about 9.0, about 10,about 10.5, about 11.0, about 11.5, about 12.0, about 12.5, about 13.0,about 13.5, about 14.0, about 14.5, about 15.0, about 15.5, about 16.0,about 16.5, about 17.0, about 17.5, about 18.0, about 18.5, about 19.0,about 19.5, about 20, about 20.5, about 21.0, about 21.5, about 22.0,about 22.5, about 23.0, about 23.5, about 24.0, about 24.5, about 25.0,about 25.5, about 26.0, about 26.5, about 27.0, about 27.5, about 28.0,about 28.5, about 29.0, or about 29.5 fold inclusive of all values andsubranges therebetween.

In certain embodiments, L-2,3-diaminopropionic acid may be conjugated tothe C-terminal of the CPP sequences in Table 4 to facilitate conjugationto the L. In other embodiments, the C-terminal amino acid of the CPPsequences in listed in Table 4 may be substituted withL-2,3-diaminopropionic acid to facilitate conjugation to the L.

Additionally, the CPP used in the polypeptide conjugates and methodsdescribed herein can include any sequence disclosed in: U.S. applicationSer. No. 15/312,878; U.S. application Ser. No. 15/360,719; U.S. App. No.62/438,141, and U.S. App. No. 62/507,483, each of which is incorporatedby reference in its entirety for all purposes.

Linker

In various embodiments, the polypeptides disclosed herein comprise alinker (“L”). The L may be any appropriate moiety which is capable offorming a covalent bond to the Xm, CPP, or a combination thereof, toform the bicyclic peptides of the present disclosure. In certainembodiments, the L is a pharmaceutically acceptable moiety. In someembodiments, the L is any appropriate trivalent radical.

In some embodiments, the L is may be an alkylene, alkenylene,alkynylene, aryl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl,heteroaryl, or N-alkyl, each of which can be optionally substituted asdefined herein.

“Alkylene” or “alkylene chain” refers to a fully saturated, straight orbranched trivalent hydrocarbon chain radical, having from one to fortycarbon atoms. Non-limiting examples of C₂-C₄₀ alkylene include ethylene,propylene, n-butylene, ethenylene, propenylene, n-butenylene,propynylene, n-butynylene, and the like. Unless stated otherwisespecifically in the specification, an alkylene chain can be optionallysubstituted as described herein.

“Alkenylene” or “alkenylene chain” refers to a straight or branchedtrivalent hydrocarbon chain radical, having from two to forty carbonatoms, and having one or more carbon-carbon double bonds. Non-limitingexamples of C₂-C₄₀ alkenylene include ethene, propene, butene, and thelike. Unless stated otherwise specifically in the specification, analkenylene chain can be optionally substituted.

“Alkynylene” or “alkynylene chain” refers to a straight or branchedtrivalent hydrocarbon chain radical, having from two to forty carbonatoms, and having one or more carbon-carbon triple bonds. Non-limitingexamples of C₂-C₄₀ alkynylene include ethynylene, propargylene and thelike. Unless stated otherwise specifically in the specification, analkynylene chain can be optionally substituted.

“Aryl” refers to a hydrocarbon ring system trivalent radical comprisinghydrogen, 6 to 40 carbon atoms and at least one aromatic ring. Forpurposes of this invention, the aryl trivalent radical can be amonocyclic, bicyclic, tricyclic or tetracyclic ring system, which caninclude fused or bridged ring systems. Aryl trivalent radicals include,but are not limited to, aryl trivalent radicals derived fromaceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene,benzene, chrysene, fluoranthene, fluorene, as-indacene, s-indacene,indane, indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene,and triphenylene. Unless stated otherwise specifically in thespecification, an aryl group can be optionally substituted.

“Cycloalkyl” refers to a stable non-aromatic monocyclic or polycyclicfully saturated hydrocarbon trivalent radical having from 3 to 40 carbonatoms and at least one ring, wherein the ring consists solely of carbonand hydrogen atoms, which can include fused or bridged ring systems.Monocyclic cycloalkyl trivalent radicals include, for example,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, andcyclooctyl. Polycyclic cycloalkyl trivalent radicals include, forexample, adamantyl, norbornyl, decalinyl,7,7-dimethyl-bicyclo[2.2.1]heptanyl, and the like. Unless otherwisestated specifically in the specification, a cycloalkyl group can beoptionally substituted.

“Cycloalkenyl” refers to a stable non-aromatic monocyclic or polycyclichydrocarbon trivalent radical having from 3 to 40 carbon atoms, at leastone ring having, and one or more carbon-carbon double bonds, wherein thering consists solely of carbon and hydrogen atoms, which can includefused or bridged ring systems. Monocyclic cycloalkenyl radicals include,for example, cyclopentenyl, cyclohexenyl, cycloheptenyl, cycloctenyl,and the like. Polycyclic cycloalkenyl radicals include, for example,bicyclo[2.2.1]hept-2-enyl and the like. Unless otherwise statedspecifically in the specification, a cycloalkenyl group can beoptionally substituted.

“Cycloalkynyl” refers to a stable non-aromatic monocyclic or polycyclichydrocarbon trivalent radical having from 3 to 40 carbon atoms, at leastone ring having, and one or more carbon-carbon triple bonds, wherein thering consists solely of carbon and hydrogen atoms, which can includefused or bridged ring systems. Monocyclic cycloalkynyl radicals include,for example, cycloheptynyl, cyclooctynyl, and the like. Unless otherwisestated specifically in the specification, a cycloalkynyl group can beoptionally substituted.

“Heterocyclyl,” “heterocyclic ring” or “heterocycle” refers to a stable3- to 20-membered non-aromatic ring trivalent radical which consists oftwo to twelve carbon atoms and from one to six heteroatoms selected fromthe group consisting of nitrogen, oxygen and sulfur. Heterocyclycl orheterocyclic rings include heteroaryls as defined below. Unless statedotherwise specifically in the specification, the heterocyclyl radicalcan be a monocyclic, bicyclic, tricyclic or tetracyclic ring system,which can include fused or bridged ring systems; and the nitrogen,carbon or sulfur atoms in the heterocyclyl radical can be optionallyoxidized; the nitrogen atom can be optionally quaternized; and theheterocyclyl radical can be partially or fully saturated. Examples ofsuch heterocyclyl radicals include, but are not limited to, dioxolanyl,thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl,imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl,octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl,2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl,piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl,thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl,thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and1,1-dioxo-thiomorpholinyl. Unless stated otherwise specifically in thespecification, a heterocyclyl group can be optionally substituted.

“Heteroaryl” refers to a 5- to 20-membered ring system radicalcomprising hydrogen atoms, one to thirteen carbon atoms, one to sixheteroatoms selected from the group consisting of nitrogen, oxygen andsulfur, and at least one aromatic ring. For purposes of this invention,the heteroaryl radical can be a monocyclic, bicyclic, tricyclic ortetracyclic ring system, which can include fused or bridged ringsystems; and the nitrogen, carbon or sulfur atoms in the heteroarylradical can be optionally oxidized; the nitrogen atom can be optionallyquaternized. Examples include, but are not limited to, azepinyl,acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl,benzofuranyl, benzooxazolyl, benzothiazolyl, benzothiadiazolyl,benzo[b][1,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl,benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl,benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl(benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl,carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl,furanonyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl,isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl,isoxazolyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl,oxiranyl, 1-oxidopyridinyl, 1-oxidopyrimidinyl, 1-oxidopyrazinyl,1-oxidopyridazinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl,phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl,pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl,quinoxalinyl, quinolinyl, quinuclidinyl, isoquinolinyl,tetrahydroquinolinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl,triazinyl, and thiophenyl (i.e. thienyl). Unless stated otherwisespecifically in the specification, a heteroaryl group can be optionallysubstituted.

“N-alkyl” refers to a alkyl radical as defined above containing at leastone nitrogen and where a point of attachment of the alkyl radical to therest of the molecule is through a nitrogen atom in the N-alkyl radical.Unless stated otherwise specifically in the specification, a N-alkylgroup can be optionally substituted.

The term “substituted” used herein means any of the above groups (i.e.,alkylene, alkenylene, alkynylene, aryl, carbocyclyl, cycloalkyl,cycloalkenyl, cycloalkynyl, heterocyclyl, and/or heteroaryl) wherein atleast one hydrogen atom is replaced by a bond to a non-hydrogen atomssuch as, but not limited to: a halogen atom such as F, Cl, Br, and I; anoxygen atom in groups such as hydroxyl groups, alkoxy groups, and estergroups; a sulfur atom in groups such as thiol groups, thioalkyl groups,sulfone groups, sulfonyl groups, and sulfoxide groups; a nitrogen atomin groups such as amines, amides, alkylamines, dialkylamines,arylamines, alkylarylamines, diarylamines, N-oxides, imides, andenamines; a silicon atom in groups such as trialkylsilyl groups,dialkylarylsilyl groups, alkyldiarylsilyl groups, and triarylsilylgroups; and other heteroatoms in various other groups. “Substituted”also means any of the above groups in which one or more hydrogen atomsare replaced by a higher-order bond (e.g., a double- or triple-bond) toa heteroatom such as oxygen in oxo, carbonyl, carboxyl, and estergroups; and nitrogen in groups such as imines, oximes, hydrazones, andnitriles. For example, “substituted” includes any of the above groups inwhich one or more hydrogen atoms are replaced with —NR_(g)R_(h),—NR_(g)C(═O)R_(h), —NR_(g)C(═O)NR_(g)R_(h), —NR_(g)C(═O)OR_(h),—NR_(g)SO₂R_(h), —OC(═O)NR_(g)R_(h), —OR_(g), —SR_(g), —SOR_(g),—SO₂R_(g), —OSO₂R_(g), —SO₂OR_(g), ═NSO₂R_(g), and —SO₂NR_(g)R_(h).“Substituted also means any of the above groups in which one or morehydrogen atoms are replaced with —C(═O)R_(g), —C(═O)OR_(g),—C(═O)NR_(g)R_(h), —CH₂SO₂R_(g), —CH₂SO₂NR_(g)R_(h). In the foregoing,R_(g) and R_(h) are the same or different and independently hydrogen,alkyl, alkenyl, alkynyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl,cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkylalkyl, haloalkyl,haloalkenyl, haloalkynyl, heterocyclyl, N-heterocyclyl,heterocyclylalkyl, heteroaryl, N-heteroaryl and/or heteroarylalkyl.“Substituted” further means any of the above groups in which one or morehydrogen atoms are replaced by a bond to an amino, cyano, hydroxyl,imino, nitro, oxo, thioxo, halo, alkyl, alkenyl, alkynyl, alkoxy,alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkenyl,cycloalkynyl, cycloalkylalkyl, haloalkyl, haloalkenyl, haloalkynyl,heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl,N-heteroaryl and/or heteroarylalkyl group. In addition, each of theforegoing substituents can also be optionally substituted with one ormore of the above substituents. Further, those skilled in the art willrecognize that “substituted” also encompasses instances in which one ormore hydrogen atoms on any of the above groups are replaced by asubstituent listed in this paragraph, and the substituent forms acovalent bond with the CPP or the Xm. For example, in certainembodiments, any of the above groups (i.e., alkylene, alkenylene,alkynylene, aryl, carbocyclyl, cycloalkyl, cycloalkenyl, cycloalkynyl,heterocyclyl, and/or heteroaryl) can be substituted at one or morepositions with a carboxylic acid (i.e., —C(═O)OH) which forms an amidebond with an amino group in the CPP and/or Xm (e.g., the N-terminus ofthe CPP or the Xm, or an amino group on a side chain of an amino acid inthe CPP and/or Xm).

In some embodiments, the L is an aryl ring, which is independentlysubstituted at three separate locations on the aryl ring. In certainembodiments, the aryl ring is a phenyl ring.

In some embodiments, the L forms a covalent bond with an amino acid inthe Xm and/or the CPP. The resulting moiety, when L forms a bond to Xmand/or the CPP, to form the bicyclic polypeptides described herein, arereferred to as R1, R2, and R3. In other embodiments, the L forms acovalent bond with the N- or C-terminus of an amino acid in the Xmand/or the CPP, or the side chain of an amino acid in the Xm and/or theCPP.

In some embodiments, each of R1, R2, and R3 are independently selectedform an amide, an ester, and triazole, and combinations thereof. Infurther embodiments, each of R1, R2, and R3 are and an amide.

In certain embodiments, the bicyclic polypeptide has the structure ofFormula 1A, 1B, 1C. or 1D, wherein each AA_(L), when present, is,independently, selected from a D or L amino acid.

Each AA_(L) may be a natural or non-natural amino acid as describedabove. In particular embodiments, p is one and AA_(L) is Dap.

In some embodiments, the precursor to L (i.e., the moiety before L formsone or more covalent bonds to the Xm and/or CPP) is L-(C(O)OH)₃. In somesuch embodiments, the precursor to L has a structure according toFormula 4:

wherein a, b, and c are independently selected from a number from 0 to10.

In various embodiments, the disclosure provides for bicyclicpolypeptides according to Formula 5A or 5B:

-   -   wherein:    -   the CPP comprises a sequence according to Formula 2:        (AA_(u))_(m)-AA₁-AA₂-AA₃-AA₄-(AA_(z))_(n)  2        -   wherein:            -   each of AA₁, AA₂, AA₃, and AA₄, are independently                selected from a D or L amino acid,            -   each of AA_(u) and AA_(z) at each instance and when                present, are independently selected from a D or L amino                acid, and            -   m and n are independently selected from a number from 0                to 6; and        -   wherein:            -   at least two of AA_(u), when present, AA₁, AA₂, AA₃,                AA₄, and AA_(z) when present, are independently                arginine, and            -   at least two of AA_(u), when present, AA₁, AA₂, AA₃,                AA₄, and AA_(z), when present, are independently a                hydrophobic amino acid;    -   Xm is a peptide sequence is a 3-10 amino acid sequence        comprising one or more amino acids selected from G, g, W, w, I,        i, Y, y, A, and a (SEQ ID NO: 64);    -   L is a linker moiety; and    -   each of R1, R2, and R3 are independently, a bonding moiety,        -   wherein the bonding moiety is formed when Xm, CPP, or a            combination thereof, covalently bind to L to form the            bicyclic polypeptide.

In various embodiments, the disclosure provides for bicyclicpolypeptides according to Formula 5C or 5D:

wherein:

the CPP comprises a sequence according to Formula 2:(AA_(u))_(m)-AA₁-AA₂-AA₃-AA₄-(AA_(z))_(n)  2

-   -   wherein:        -   each of AA₁, AA₂, AA₃, and AA₄, are independently selected            from a D or L amino acid,        -   each of AA_(u) and AA_(z), at each instance and when            present, are independently selected from a D or L amino            acid, and        -   m and n are independently selected from a number from 0 to            6; and    -   wherein:        -   at least two of AA_(u), when present, AA₁, AA₂, AA₃, AA₄,            and AA_(z), when present, are independently arginine, and    -   at least two of AA_(u), when present, AA₁, AA₂, AA₃, AA₄, and        AA_(z) when present, are independently a hydrophobic amino acid;    -   Xm is a peptide sequence is a 3-10 amino acid sequence        comprising one or more amino acids selected from G, g, W, w, I,        i, Y,y, A, and a (SEQ ID NO: 64);    -   AA_(L) at each instance is an amino acid;    -   p is selected from a number from 0 to 3 (e.g., 0, 1, 2, or 3);    -   L is a linker moiety; and    -   each of R1, R2, and R3 are independently, a bonding moiety,        -   wherein the bonding moiety is formed when Xm, CPP, or a            combination thereof, covalently bind to L to form the            bicyclic polypeptide.

In further embodiments of the invention, the CPP of the bicyclicpolypeptide of Formula 5A-5D comprises a sequence according to Formula3A-D:

wherein:

-   -   each of AA_(H1) and AA_(H2) are independently a hydrophobic        amino acid;    -   at each instance and when present, each of AA_(U) and AA_(Z) are        independently any amino acid; and    -   m and n are independently selected from a number from 0 to 6.

In still further embodiments of the invention the Xm peptide sequence ofthe bicyclic polypeptide of Formulae 1A-1D and 5A-5D is a 3-7 amino acidsequence comprising a sequence selected from WI, IW, Wi, iW, wI, Iw, iw,wi.

In yet further embodiments of the invention, the Xm peptide sequence ofthe bicyclic polypeptide of Formulae 1A-1D and 5A-5D is a 4-7 amino acidsequence comprising a sequence selected from the group consisting of:GWIY (SEQ ID NO:1); GWIYA (SEQ ID NO:2); GWIYa (SEQ ID NO: 50); AGWIY(SEQ ID NO:3); aGWIY (SEQ ID NO: 51); AWIYA (SEQ ID NO:4); GAIYA (SEQ IDNO:5); GWAYA (SEQ ID NO:6); GWIAA (SEQ ID NO:7); GWIYA (SEQ ID NO:8);GAIAA (SEQ ID NO:9); and GAAAA (SEQ ID NO:10), and the inverse of suchsequences (SEQ ID NOs: 52-63).

Methods of Treatment

In some embodiments, the polypeptides disclosed herein inhibit theNEMO-IKKα/β interaction by at least about 10%, e.g., about 15%, about20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%,about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%,about 96%, about 97%, about 98%, about 99%, and about 100%, inclusive ofall values and subranges therebetween.

As used herein, the terms “inhibit,” inhibited,” “inhibition,”“inhibiting” or other derivations or variations thereof refer to theactivity of a particular enzyme which is reduced by using an inhibitor.In some embodiments, “inhibition” can refer to complete loss of functionof an enzyme or a reduction of the activity of the enzyme (e.g., byabout 1% or more). The level of reduction is compared to a comparablehost cell of the same species which is not treated by the inhibitor.

In some embodiments, the polypeptides disclosed herein have an IC₅₀ ofabout 10 μM or less when measured for the NEMO-IKKα/β interaction, e.g.,about 9 μM, about 8 μM, about 7 μM, about 6 μM, about 5 μM, about 4 μM,about 3 μM, about 2 μM, about 1 μM, about 0.9 μM, about 0.8 μM, about0.7 μM, about 0.6 μM, about 0.5 μM, about 0.4 μM, about 0.3 μM, about0.2 μM, about 0.1 μM, about 0.09 μM, about 0.08 μM, about 0.07 μM, about0.06 μM, about 0.05 μM, about 0.04 μM, about 0.03 μM, about 0.02 μM, orabout 0.01 μM or less, inclusive of all values and subrangestherebetween. In particular embodiments, polypeptides have an IC50 ofabout 1.0 μM or less when measured for the NEMO-IKKα/β interaction.

Methods of Making

The polypeptide conjugates described herein can be prepared in a varietyof ways known to one skilled in the art of organic synthesis orvariations thereon as appreciated by those skilled in the art. Thecompounds described herein can be prepared from readily availablestarting materials. Optimum reaction conditions can vary with theparticular reactants or solvents used, but such conditions can bedetermined by one skilled in the art.

Variations on the compounds described herein include the addition,subtraction, or movement of the various constituents as described foreach compound. Similarly, when one or more chiral centers are present ina molecule, the chirality of the molecule can be changed. Additionally,compound synthesis can involve the protection and deprotection ofvarious chemical groups. The use of protection and deprotection, and theselection of appropriate protecting groups can be determined by oneskilled in the art. The chemistry of protecting groups can be found, forexample, in Wuts and Greene, Protective Groups in Organic Synthesis, 4thEd., Wiley & Sons, 2006, which is incorporated herein by reference inits entirety.

The starting materials and reagents used in preparing the disclosedcompounds and compositions are either available from commercialsuppliers such as Aldrich Chemical Co., (Milwaukee, Wis.), AcrosOrganics (Morris Plains, N.J.), Fisher Scientific (Pittsburgh, Pa.),Sigma (St. Louis, Mo.), Pfizer (New York, N.Y.), GlaxoSmithKline(Raleigh, N.C.), Merck (Whitehouse Station, N.J.), Johnson & Johnson(New Brunswick, N.J.), Aventis (Bridgewater, N.J.), AstraZeneca(Wilmington, Del.), Novartis (Basel, Switzerland), Wyeth (Madison,N.J.), Bristol-Myers-Squibb (New York, N.Y.), Roche (Basel,Switzerland), Lilly (Indianapolis, Ind.), Abbott (Abbott Park, Ill.),Schering Plough (Kenilworth, N.J.), or Boehringer Ingelheim (Ingelheim,Germany), or are prepared by methods known to those skilled in the artfollowing procedures set forth in references such as Fieser and Fieser'sReagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons,1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 andSupplementals (Elsevier Science Publishers, 1989); Organic Reactions,Volumes 1-40 (John Wiley and Sons, 1991); March's Advanced OrganicChemistry, (John Wiley and Sons, 4th Edition); and Larock'sComprehensive Organic Transformations (VCH Publishers Inc., 1989). Othermaterials, such as the pharmaceutical carriers disclosed herein can beobtained from commercial sources.

Reactions to produce the compounds described herein can be carried outin solvents, which can be selected by one of skill in the art of organicsynthesis. Solvents can be substantially nonreactive with the startingmaterials (reactants), the intermediates, or products under theconditions at which the reactions are carried out, i.e., temperature andpressure. Reactions can be carried out in one solvent or a mixture ofmore than one solvent. Product or intermediate formation can bemonitored according to any suitable method known in the art. Forexample, product formation can be monitored by spectroscopic means, suchas nuclear magnetic resonance spectroscopy (e.g., ¹H or ¹³C) infraredspectroscopy, spectrophotometry (e.g., UV-visible), or massspectrometry, or by chromatography such as high-performance liquidchromatography (HPLC) or thin layer chromatography.

The disclosed compounds can be prepared by solid phase peptide synthesiswherein the amino acid α-N-terminal is protected by an acid or baseprotecting group. Such protecting groups should have the properties ofbeing stable to the conditions of peptide linkage formation while beingreadily removable without destruction of the growing peptide chain orracemization of any of the chiral centers contained therein. Suitableprotecting groups are 9-fluorenylmethyloxycarbonyl (Fmoc),t-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz),biphenylisopropyloxycarbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl,α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, o-nitrophenylsulfenyl,2-cyano-t-butyloxycarbonyl, and the like. The9-fluorenylmethyloxycarbonyl (Fmoc) protecting group is particularlypreferred for the synthesis of the disclosed compounds. Other preferredside chain protecting groups are, for side chain amino groups likelysine and arginine, 2,2,5,7,8-pentamethylchroman-6-sulfonyl (pmc),nitro, p-toluenesulfonyl, 4-methoxybenzene-sulfonyl, Cbz, Boc, andadamantyloxycarbonyl; for tyrosine, benzyl, o-bromobenzyloxy-carbonyl,2,6-dichlorobenzyl, isopropyl, t-butyl (t-Bu), cyclohexyl, cyclopenyland acetyl (Ac); for serine, t-butyl, benzyl and tetrahydropyranyl; forhistidine, trityl, benzyl, Cbz, p-toluenesulfonyl and 2,4-dinitrophenyl;for tryptophan, formyl; for asparticacid and glutamic acid, benzyl andt-butyl and for cysteine, triphenylmethyl (trityl). In the solid phasepeptide synthesis method, the α-C-terminal amino acid is attached to asuitable solid support or resin. Suitable solid supports useful for theabove synthesis are those materials which are inert to the reagents andreaction conditions of the stepwise condensation-deprotection reactions,as well as being insoluble in the media used. Solid supports forsynthesis of α-C-terminal carboxy peptides is4-hydroxymethylphenoxymethyl-copoly (styrene-1% divinylbenzene) or4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)phenoxyacetamidoethyl resinavailable from Applied Biosystems (Foster City, Calif.). Theα-C-terminal amino acid is coupled to the resin by means ofN,N′-dicyclohexylcarbodiimide (DCC), N,N′-diisopropylcarbodiimide (DIC)or O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluroniumhexafluorophosphate(HBTU), with or without 4-dimethylaminopyridine (DMAP),1-hydroxybenzotriazole (HOBT),benzotriazol-1-yloxy-tris(dimethylamino)phosphoniumhexafluorophosphate(BOP) or bis(2-oxo-3-oxazolidinyl)phosphine chloride (BOPCl), mediatedcoupling for from about 1 to about 24 hours at a temperature of between10° C. and 50° C. in a solvent such as dichloromethane or DMF. When thesolid support is4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy-acetamidoethyl resin,the Fmoc group is cleaved with a secondary amine, preferably piperidine,prior to coupling with the α-C-terminal amino acid as described above.One method for coupling to the deprotected 4(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy-acetamidoethyl resin isO-benzotriazol-1-yl-N,N,N′,N′-tetramethyluroniumhexafluorophosphate(HBTU, 1 equiv.) and 1-hydroxybenzotriazole (HOBT, 1 equiv.) in DMF. Thecoupling of successive protected amino acids can be carried out in anautomatic polypeptide synthesizer. In one example, the α-N-terminal inthe amino acids of the growing peptide chain are protected with Fmoc.The removal of the Fmoc protecting group from the α-N-terminal side ofthe growing peptide is accomplished by treatment with a secondary amine,preferably piperidine. Each protected amino acid is then introduced inabout 3-fold molar excess, and the coupling is preferably carried out inDMF. The coupling agent can beO-benzotriazol-1-yl-N,N,N′,N′-tetramethyluroniumhexafluorophosphate(HBTU, 1 equiv.) and 1-hydroxybenzotriazole (HOBT, 1 equiv.). At the endof the solid phase synthesis, the polypeptide is removed from the resinand deprotected, either in successively or in a single operation.Removal of the polypeptide and deprotection can be accomplished in asingle operation by treating the resin-bound polypeptide with a cleavagereagent comprising thianisole, water, ethanedithiol and trifluoroaceticacid. In cases wherein the α-C-terminal of the polypeptide is analkylamide, the resin is cleaved by aminolysis with an alkylamineAlternatively, the peptide can be removed by transesterification, e.g.with methanol, followed by aminolysis or by direct transamidation. Theprotected peptide can be purified at this point or taken to the nextstep directly. The removal of the side chain protecting groups can beaccomplished using the cleavage cocktail described above. The fullydeprotected peptide can be purified by a sequence of chromatographicsteps employing any or all of the following types: ion exchange on aweakly basic resin (acetate form); hydrophobic adsorption chromatographyon underivitized polystyrene-divinylbenzene (for example, AmberliteXAD); silica gel adsorption chromatography; ion exchange chromatographyon carboxymethylcellulose; partition chromatography, e.g. on SephadexG-25, LH-20 or countercurrent distribution; high performance liquidchromatography (HPLC), especially reverse-phase HPLC on octyl- oroctadecylsilyl-silica bonded phase column packing.

Methods of Administration

In vivo application of the disclosed polypeptide conjugates, andcompositions containing them, can be accomplished by any suitable methodand technique presently or prospectively known to those skilled in theart. For example, the disclosed compounds can be formulated in aphysiologically- or pharmaceutically-acceptable form and administered byany suitable route known in the art including, for example, oral,intranasal, and parenteral routes of administration. As used herein, theterm parenteral includes subcutaneous, intradermal, intravenous,intramuscular, intraperitoneal, and intrasternal administration, such asby injection. Administration of the disclosed compounds or compositionscan be a single administration, or at continuous or distinct intervalsas can be readily determined by a person skilled in the art.

The compounds disclosed herein, and compositions comprising them, canalso be administered utilizing liposome technology, slow releasecapsules, implantable pumps, and biodegradable containers. Thesedelivery methods can, advantageously, provide a uniform dosage over anextended period of time. The compounds can also be administered in theirsalt derivative forms or crystalline forms.

The compounds disclosed herein can be formulated according to knownmethods for preparing pharmaceutically acceptable compositions.Formulations are described in detail in a number of sources which arewell known and readily available to those skilled in the art. Forexample, Remington's Pharmaceutical Science by E. W. Martin (1995)describes formulations that can be used in connection with the disclosedmethods. In general, the compounds disclosed herein can be formulatedsuch that an effective amount of the compound is combined with asuitable carrier in order to facilitate effective administration of thecompound. The compositions used can also be in a variety of forms. Theseinclude, for example, solid, semi-solid, and liquid dosage forms, suchas tablets, pills, powders, liquid solutions or suspension,suppositories, injectable and infusible solutions, and sprays. Thepreferred form depends on the intended mode of administration andtherapeutic application. The compositions also preferably includeconventional pharmaceutically-acceptable carriers and diluents which areknown to those skilled in the art. Examples of carriers or diluents foruse with the compounds include ethanol, dimethyl sulfoxide, glycerol,alumina, starch, saline, and equivalent carriers and diluents. Toprovide for the administration of such dosages for the desiredtherapeutic treatment, compositions disclosed herein can advantageouslycomprise between about 0.1% and 100% by weight of the total of one ormore of the subject compounds based on the weight of the totalcomposition including carrier or diluent.

Formulations suitable for administration include, for example, aqueoussterile injection solutions, which can contain antioxidants, buffers,bacteriostats, and solutes that render the formulation isotonic with theblood of the intended recipient; and aqueous and nonaqueous sterilesuspensions, which can include suspending agents and thickening agents.The formulations can be presented in unit-dose or multi-dose containers,for example sealed ampoules and vials, and can be stored in a freezedried (lyophilized) condition requiring only the condition of thesterile liquid carrier, for example, water for injections, prior to use.Extemporaneous injection solutions and suspensions can be prepared fromsterile powder, granules, tablets, etc. It should be understood that inaddition to the ingredients particularly mentioned above, thecompositions disclosed herein can include other agents conventional inthe art having regard to the type of formulation in question.

Compounds disclosed herein, and compositions comprising them, can bedelivered to a cell either through direct contact with the cell or via acarrier means. Carrier means for delivering compounds and compositionsto cells are known in the art and include, for example, encapsulatingthe composition in a liposome moiety. Another means for delivery ofcompounds and compositions disclosed herein to a cell comprisesattaching the compounds to a protein or nucleic acid that is targetedfor delivery to the target cell. U.S. Pat. No. 6,960,648 and U.S.Application Publication Nos. 20030032594 and 20020120100 disclose aminoacid sequences that can be coupled to another composition and thatallows the composition to be translocated across biological membranes.U.S. Application Publication No. 20020035243 also describes compositionsfor transporting biological moieties across cell membranes forintracellular delivery. Compounds can also be incorporated intopolymers, examples of which include poly (D-L lactide-co-glycolide)polymer for intracranial tumors; poly[bis(p-carboxyphenoxy) propane:sebacic acid] in a 20:80 molar ratio (as used in GLIADEL); chondroitin;chitin; and chitosan.

Compounds and compositions disclosed herein, including pharmaceuticallyacceptable salts or prodrugs thereof, can be administered intravenously,intramuscularly, or intraperitoneally by infusion or injection.Solutions of the active agent or its salts can be prepared in water,optionally mixed with a nontoxic surfactant. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, triacetin, andmixtures thereof and in oils. Under ordinary conditions of storage anduse, these preparations can contain a preservative to prevent the growthof microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient, which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. The ultimatedosage form should be sterile, fluid and stable under the conditions ofmanufacture and storage. The liquid carrier or vehicle can be a solventor liquid dispersion medium comprising, for example, water, ethanol, apolyol (for example, glycerol, propylene glycol, liquid polyethyleneglycols, and the like), vegetable oils, nontoxic glyceryl esters, andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the formation of liposomes, by the maintenance of therequired particle size in the case of dispersions or by the use ofsurfactants. Optionally, the prevention of the action of microorganismscan be brought about by various other antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, sorbic acid,thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the inclusion of agents that delay absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating a compoundand/or agent disclosed herein in the required amount in the appropriatesolvent with various other ingredients enumerated above, as required,followed by filter sterilization. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and the freeze-drying techniques, whichyield a powder of the active ingredient plus any additional desiredingredient present in the previously sterile-filtered solutions.

Useful dosages of the compounds and agents and pharmaceuticalcompositions disclosed herein can be determined by comparing their invitro activity, and in vivo activity in animal models. Methods for theextrapolation of effective dosages in mice, and other animals, to humansare known to the art.

The dosage ranges for the administration of the compositions are thoselarge enough to produce the desired effect in which the symptoms ordisorder are affected. The dosage should not be so large as to causeadverse side effects, such as unwanted cross-reactions, anaphylacticreactions, and the like. Generally, the dosage will vary with the age,condition, sex and extent of the disease in the patient and can bedetermined by one of skill in the art. The dosage can be adjusted by theindividual physician in the event of any counterindications. Dosage canvary, and can be administered in one or more dose administrations daily,for one or several days.

Also disclosed are pharmaceutical compositions that comprise a compounddisclosed herein in combination with a pharmaceutically acceptablecarrier. Pharmaceutical compositions adapted for oral, topical orparenteral administration, comprising an amount of a compound constitutea preferred aspect. The dose administered to a patient, particularly ahuman, should be sufficient to achieve a therapeutic response in thepatient over a reasonable time frame, without lethal toxicity, andpreferably causing no more than an acceptable level of side effects ormorbidity. One skilled in the art will recognize that dosage will dependupon a variety of factors including the condition (health) of thesubject, the body weight of the subject, kind of concurrent treatment,if any, frequency of treatment, therapeutic ratio, as well as theseverity and stage of the pathological condition.

Also disclosed are kits that comprise a compound disclosed herein in oneor more containers. The disclosed kits can optionally includepharmaceutically acceptable carriers and/or diluents. In one embodiment,a kit includes one or more other components, adjuncts, or adjuvants asdescribed herein. In another embodiment, a kit includes one or moreanti-cancer agents, such as those agents described herein. In oneembodiment, a kit includes instructions or packaging materials thatdescribe how to administer a compound or composition of the kit.Containers of the kit can be of any suitable material, e.g., glass,plastic, metal, etc., and of any suitable size, shape, or configuration.In one embodiment, a compound and/or agent disclosed herein is providedin the kit as a solid, such as a tablet, pill, or powder form. Inanother embodiment, a compound and/or agent disclosed herein is providedin the kit as a liquid or solution. In one embodiment, the kit comprisesan ampoule or syringe containing a compound and/or agent disclosedherein in liquid or solution form.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

EXAMPLES Example 1. Design and Synthesis of Combinatorial Library ofCell-Permeable Bicyclic Peptides

A bicyclic peptide library featuring random peptide sequences of 3-6residues in the first ring (A ring) and 12 different CPP sequences inthe second ring (B ring) was designed and prepared (FIG. 1). The peptidelibrary was synthesized on 2 g of TentaGel S NH2 resin (130 μm) bymodifying a previously reported protocol disclosed in Lian et al. (Am.Chem. Soc. 2013, 135, 11990-11995), which is incorporated by referenceherein in its entirety. Library screening was also performed accordingto previously established protocols in Lian et al. (Am. Chem. Soc. 2013,135, 11990-11995). In brief, approximately 600 mg of the bicyclicpeptide library was swollen in DCM and washed extensively with DMF,ddH₂O and finally incubated overnight at 4° C. in 1.0 mL of blockingbuffer (30 mM sodium phosphate, pH 7.4, 150 mM NaCl, 0.05% Tween 20, 3%BSA and 0.1% gelatin). The solution was drained and the resin wasresuspended in blocking buffer containing 1.0 μM biotinylated GST-NEMOfor 4 hours at 4° C. Unbound NEMO was washed away with blocking bufferand the beads were resuspended in 10 mL of blocking buffer. Twenty μL ofM280 streptavidin-coated Dynabeads (Invitrogen) was added to thesolution and allowed to incubate on a rotary wheel for 1 hour at 4° C.The magnetic beads were isolated from the bulk by using a TA Dynal MPC-1magnetic particle concentrator (Invitrogen). Hit beads were transferredto a Bio-Spin column (0.8 mL, BioRad) and incubated in blocking buffercontaining 1.0 μM biotinylated GST-NEMO for 4 hours at 4° C. Thesolution was drained and the resin was washed with blocking buffer toremove unbound protein. The resin was resuspended in 1 mL of blockingbuffer and streptavidin-alkaline phosphatase (SA-AP) conjugate was addedto the tube (1 mg/mL final concentration). After 10 minutes at 4° C. thesolution was drained and the beads were quickly washed with 1 mL ofblocking buffer (3×) and 1 mL of staining buffer (30 mM Tris pH 8.5, 100mM NaCl, 5 mM MgCl₂, and 20 μM ZnCl₂) (3×). The resin was resuspended in1.5 mL of staining buffer in a petri dish and 150 μL of a5-bromo-4-chloro-3-indolyl-phosphate (BCIP) solution (5 mg/mL) wasadded. After 30 minutes, 50 μL of 1 M HCl was added to quench thereaction and the intensely turquoise positive beads were isolated undera dissecting microscope. The sequences of hit beads were determinedusing partial Edman degradation-mass spectrometry (PED-MS) as previouslydescribed in Thakkar et al. (Anal. Chem. 2006, 78, 5935-5939), which isincorporated by reference herein in its entirety.

The resulting peptide sequences in the A ring were constructed throughcombinatorial synthesis using a set of 24 proteinogenic and unnaturalamino acids (e.g., D-amino acids). The 12 CPP sequences consisted ofdifferent combinations of two or three aromatic hydrophobic residues (L-or D-Phe and L- or D-naphthylalanine) and three or four L- or D-arginineresidues and were prepared by parallel synthesis. Because the NEMOsurface near the IKK-binding site is negatively charged (vide infra), inaddition to ensuring cell penetration, some of the positively chargedCPP sequences might also interact electrostatically with the negativelycharged NEMO surface. The bicyclic library has a theoretical diversityof 2.4×10⁹ and was synthesized on 130-TentaGel beads in the one bead-twocompound (OBTC) format, with each bead displaying a unique bicyclicpeptide on its surface layer and a linear peptide of identical sequencein its inner layer as an encoding tag. The library design also includeda propargylglycine-β-alanine-hydroxylmethylbenzoyl ester (Pra-B-Hmb)linker, which was intended for selective on-bead fluorescent labelingand release of the bicyclic peptide for an additional round ofin-solution screening, although the latter turned out to be unnecessary.

Approximately 600 mg of the library (about 600,000 different compounds)was screened for binding to NEMO in two different rounds as detailedunder Methods, resulting in 12 unique hit sequences. Ten of these 12peptides were resynthesized and tested for inhibition of the NEMO-IKKβinteraction by using a homogenous time-resolved fluorescence (HTRF)assay. All 10 peptides inhibited the NEMO-IKKβ interaction with IC50values of 3.4-28 μM. The three most potent compounds were labelled withfluorescein and their cellular uptake into HeLa cells was quantitated byflow cytometry analysis. All three peptides were cell-permeable and hadsimilar cell entry efficiency. Peptide 2 (Table 5) was selected forfurther optimization, because it showed the highest potency in the HTRFassay, with an IC50 value of 3.4 μM (FIG. 2A).

For flow cytometry analysis, HeLa cells were seeded into 12-well plates(1.5×10⁵ cells per well) 24 hours before treatment. The next day, 5 μMFITC-labeled peptide in DMEM containing 10% FBS was added to each welland allowed to incubate at 37° c. for 2 hours. After compound treatment,the cells were washed with DPBS, detached from the plate with trypsin(0.25%), diluted in DMEM containing 10% FBS and pelleted at 250 g for 5min. This washing process was repeated once to remove any free compoundand left over trypsin. The washed pellet was resuspended in DPBS with 1%FBS and analyzed on a BD LSR II flow cytometer. Data presented were themean±SD of three independent experiments.

Recombinant GST-NEMO (20 nM), biotin-IKKβKK/RR(701-745) (50 nM),streptavidin labeled with d2 acceptor (2.5 μg/mL), anti-GST monoclonalantibody labeled with Tb donor (2.5 μg/mL), and varying concentrationsof peptide (O-100 μM) were mixed in PBS containing 1 mM TCEP and 0.01%Triton X-100 (total volume 20 μL) in a 384-well plate. The plate wasincubated for 2 hOURS at room temperature to establish an equilibrium.The HTRF signals were measured on a Tecan Infinite M1000 Pro microplatereader and plotted as a function of the peptide concentration. The datawas analyzed using GraphPad Prism 6.0 and IC50 values were obtained byfitting the data to the dose-response inhibition curves. Data presentedwere the mean±SD of three independent experiments.

In general, for protein expression and purification, Escherichia coliBL21(DE3) cells were transformed with a pGEX4T3-NEMO(1-196) plasmid andgrown at 37° C. in Luria broth supplemented with 0.05 mg/mL ampicillinto an OD600 of 0.4. Expression of GST-NEMO was induced by the additionof isopropyl β-D-1-thiogalactopyranoside (150 μM final concentration).After five hours at 30° C., the cells were harvested by centrifugation.The cell pellet was suspended in 40 mL of lysis buffer (50 mM Tris-HCl,100 mM NaCl, 0.5 mM MgCl2, 5 mM O-mercaptoethanol, 0.1% Triton-X-100, pH8.0), 100 μg/mL lysozyme, 100 μL of DNAse I (New England BioLabs), and100 μL of Halt Protease Inhibitor Cocktail (EDTA-free) (ThermoScientific). This mixture was stirred at 4° C. for 30 min and brieflysonicated (2×10 s pulses). The crude lysate was centrifuged to yield aclear supernatant, which was directly loaded onto aglutathione-Sepharose 4B column (GE Healthcare). The bound protein waseluted from the column with 10 mM glutathione in 50 mM Tris-HCl (pH8.0), concentrated to 0.5 mL with the use of Amicon Ultra-15 centrifugalfilter units (MWCO 10 kDa), and dialyzed against PBS before flashfreezing.

An engineered prokaryotic expression plasmid pJCC04a, which encodes afusion protein containing an N-terminal six-histidine tag, thioredoxin,a TEV protease cleavage site, and the K703R/K704R mutant form ofIKKβC-terminal fragment (amino acids 701-745)[His-thx-IKKbKK/RR(701-745)]. His-thx-IKKbKK/RR(701-745) was similarlyexpressed in E. coli BL21 (DE3) cells and purified by affinitychromatography using a HisTrap FF column (GE Healthcare). The fusionprotein was eluted with 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 250 mMimidazole, 2 mM O-mercaptoethanol and treated with TEV protease (150units for 1 mg of fusion protein) for 16 hours at 4° C. to remove thethioredoxin (thx). The resulting protease digestion mixture was reloadedonto the HisTrap column The flow-through fraction was collected andconcentrated to about 2 mg/mL using Amicon Ultra-15 centrifugal filterunits (MWCO 10 kDa). The IKKbKK/RR(701-745) peptide was biotinylated bytreatment with a 10-fold molar excess of biotin-NHS at 4° C. overnight.The biotinylated IKKbKK/RR(701-745) was purified by reversed-phase HPLCequipped with a C18 column and stored frozen at −80° C.

In addition, a medicinal chemistry campaign was conducted to investigatethe NEMO-binding affinity and/or cell-permeability of peptide 2. Inbrief, we first modified the CPP motif in the B ring to improve cellularuptake. Addition of a 4th arginine to the CPP motif, either inside(Table 5, peptide 3) or outside the B ring (peptides 4 and 5) slightlydecreased the cellular entry efficiency. Replacement of the secondarginine with D-arginine increased the uptake efficiency by 4.2-fold(peptide 6). Therefore, FΦRrR (where Φ is L-naphthylalanine and r isD-arginine (SEQ ID NO: 66)) was chosen as the CPP motif and modified theA ring to improve the NEMO-binding affinity. Insertion of an Alaimmediately N-terminal to the internal Dap residue (with the intentionof creating an additional site of diversification) increased the bindingaffinity by 5.9-fold (IC50=1.0 μM for peptide 7; FIGS. 1 and 2A-2E).

In a fluorescence anisotropy (FA) assay, fluorescently labeled peptide 7bound directly to NEMO with a KD value of 220±80 nM, but only weakly toa panel of control proteins (KD>10 μM). The proteolytic stability ofpeptide 7 was assessed by incubation in human serum at 37° C. andquantitation of the remaining peptide as a function of time byanalytical HPLC. Peptide 7 showed minimal degradation (<1%) after 20hours of incubation. Under the same conditions, Antp-NBD (Table 5,peptide 1) was rapidly degraded with a half-life of about 20 minutes.Given its excellent proteolytic stability and respectable potency andcell permeability (163% relative to CPP1, a previously reported andrelatively efficient cyclic CPP; FIGS. 2A and 2 b), we selected peptide7 (FIG. 1) for further characterization.

To gain insight into how peptide 7 interacts with NEMO, in silicodocking of peptide 7 to the NEMO protein was performed. The all-atom 3Dstructure of peptide 7 was prepared in Maestro from the 2D structure(Schrodinger, LLC ver. 11.1) and relaxed using Polak-Ribier conjugategradient minimization to resolve steric and torsional strain introducedduring conversion. To account for the complex conformational landscapeof a bicyclic peptide, a conformational ensemble of structures wasobtained for peptide 7 using molecular dynamics. In brief, peptide 7 wasparameterized using the OPLS3 force-field and then solvated in anorthorhombic periodic box containing 1867 TIP3P water molecules. Thesystem was neutralized with the addition of three Cl⁻ ions and thensimulated for 1 ns at 303 K and 1.01 MPa, controlled using theNose-Hoover thermostat and Martyna-Tobias-Klein barostat, respectively,through the Desmond software package. Structures corresponding to every10 ps of the simulation were extracted using the Trajectory tool inMaestro. To ensure that the ensemble contains a diverse selection ofinitial compound geometries, the molecular dynamics structures werepassed through the Macrocycle Conformational Sampling tool in MacroModelusing the OPLS3 force-field. Generalized Born/Solvent Area waterelectrostatics were applied, sampling across a temperature range from300 K to 1000 K using a global low-mode search over 50 simulationcycles. Redundant conformers (defined as a heavy-atom RMSD of ≤0.75 Å)were rejected, yielding an ensemble of 105 structures spanning an energyrange of 20 kcal/mol, which were carried forward for docking.

The receptor was prepared from the reported crystal structure of a NEMOdimer (PDBID: 3V3B), by removing co-crystallized solvent andreconstructing incomplete side-chains using Prime (Schrodinger LLC). Thebinding site was defined as a 20-Å cube centered on the geometric meanof receptor residues Leu-93, Phe-97 and Val-104.Rigid-receptor/flexible-ligand docking was performed usingextra-precision mode in Glide. Ligand flexibility was accounted for byapplying a scaling factor of 0.8 to ligand atom van der Waals radii andthe generation of 100,000 poses per ligand sampled, for a theoreticaltotal of 100,000,000 poses sampled for peptide 7 during the combineddocking runs. Top scoring poses were subjected to a brief round ofenergy minimization using Embrace (Schrodinger LLC) to remove any stericclashes resulting from van der Waals' radii scaling during docking. Thefinal poses were analyzed and electrostatic potential surfaces generatedusing UCSF Chimera.

The best-scoring binding pose of peptide 7 shown in FIGS. 3A-3C. Peptide7 binds to the canonical IKK-binding site on NEMO, with the peptide Aring partially inserted into a hydrophobic groove normally occupied bythe NBD of IKKβ. The side chain of Tyr-4 is deeply inserted into a largehydrophobic pocket and excluded from the sol-vent (FIG. 3B). Ile-3interacted intimately with a shallow hydrophobic pocket on the NEMOsurface, whereas Trp-2 made surface contacts primarily through thepyrrole ring while the benzene ring was mostly solvent exposed.

The B ring (CPP ring) of peptide 7 made important interactions withNEMO. The three arginine residues of the CPP motif interactelectrostatically with the acidic patch next to the NBD binding groove(FIG. 3B). Phe-7, which is a part of the CPP motif, was completelyburied and made hydrophobic interaction with NEMO. Nal-8 was almostcompletely solvent exposed, although the naphthalene ring may makehydrophobic interactions with the NEMO surface.

To assess the validity of the molecular modeling results, we performedan “alanine scan” of peptide 7 by replacing each residue with an alanine(or D-alanine) and determining the IC50 values of the resulting peptidesagainst the NEMO-IKKβ interaction (Table 5, peptides 11-19). Consistentwith the modeling results, replacement of Trp-2, Ile-3, or Tyr-4 in theA ring with alanine significantly decreased the potency of the inhibitor(by 2- to 3-fold for peptides 12-14).

Substitution of Ala for Phe-7 (peptide 15), Nal-8 (peptide 16), or D-Argat position 10 (peptide 18) also significantly de-creased theNEMO-binding affinity. Thus, both molecular modeling and alanine-scanresults indicate that the CPP motif in the B ring also interacts withthe NEMO protein and contributes to the overall binding affinity andspecificity of peptide 7.

Based on the SAR data, we generated two negative control peptides byreplacing two (Trp-2 and Tyr-4) or three of the NEMO-binding residues inthe A ring (Trp-2, Ile-3, and Tyr-4) with Ala. The resulting peptides(peptides 20 and 21) have similar cellular entry efficiencies to peptide7, but 16- and >100-fold lower NEMO-binding affinity, respectively(FIGS. 2A and 2B and Table 5). In general, the bicyclic peptide librarystrategy developed in this work should be applicable to the discovery ofcell-permeable bicyclic peptides against numerous intracellular targets.

TABLE 5 Sequences, NEMO-binding affinities, and cell-permeability of peptide. Peptide SEQ ID IC₅₀ Permeability ID NO: Sequene(μM) (%)  1 138 RQIKIWFQNRRMKWKKGGT  >40  35 ALDWSWLQTE  2 139Tm(GWIY)Δ(FΦRRRΔ)-BBK 3.4 ± 0.6  76  3 140 Tm(GWIY)Δ(FΦRRRRΔ)-BBK1.5 ± 0.2  55  4 141 Tm(GWIY)Δ(FΦRRRΔ)-RBK 1.8 ± 0.3  71  5 142Tm(GWIY)Δ(FΦRRRΔ)-rBK 1.3 ± 0.4  71  6 143 Tm(GWIY)Δ(FΦRrRΔ)-BBK5.9 ± 0.6 322  7 144 Tm(GWIYA)Δ(FΦRrRΔ)-BBK 1.0 ± 0.1 163  8 145Tm(GWIYa)Δ(FΦRrRΔ)-BBK 2.9 ± 0.1  9 146 Tm(AGWIY)Δ(FΦRrRΔ)-BBK 2.2 ± 0.110 147 Tm(aGWIY)Δ(FΦRrRΔ)-BBK 2.3 ± 0.3 11 148 Tm(AWIYA)Δ(FΦRrRΔ)-BBK1.2 ± 0.2 12 149 Tm(GAIYA)Δ(FΦRrRΔ)-BBK 3.0 ± 0.3 13 150Tm(GWAYA)Δ(FΦRrRΔ)-BBK 1.7 ± 0.1 14 151 Tm(GWIAA)Δ(FΦRrRΔ)-BBK 2.8 ± 0.315 152 Tm(GWIYA)Δ(AΦRrRΔ)-BBK 1.9 ± 0.2 16 153 Tm(GWIYA)Δ(FARrRΔ)-BBK2.8 ± 0.2 17 154 Tm(GWIYA)Δ(FΦArRΔ)-BBK 1.1 ± 0.3 18 155Tm(GWIYA)Δ(FΦRaRΔ)-BBK 2.1 ± 0.2 19 156 Tm(GWIYA)Δ(FΦRrRΔ)-BBK 1.2 ± 0.220 157 Tm(GAIAA)Δ(FΦRrRΔ)-BBK  16 ± 2 176 21 158Tm(GAAAA)Δ(FΦRrRΔ)-BBK >100 110 Tm, trimesic acid; Δ,L-2,3-diaminopropionic acid, Φ, L-2-naphthylalanine; B, L-β-alanine; r,D-arginine. See FIG. S₁ for detailed structures. Cell-permeabilityvalues are relative to that of CPP₁ (100%).

Example 2. Inhibition of NF-κB Signaling by Bicyclic Peptidyl Inhibitors

The ability of the bicyclic peptidyl inhibitors to inhibit NF-κBSignaling was analyzed. The ability of peptide 7 to enter the cell andblock the intracellular NEMO-IKK interaction was assessed by usingHEK293(Luc) cells, which harbor a luciferase gene under thetranscriptional control of NF-κB. For the NF-κB Luciferase Assays,culture media was exchanged for DMEM containing 10% FBS and 1%penicillin/streptomycin the day before seeding to remove hygromycin B.HEK293(Luc) cells were seeded in 50 μL of assay medium (DMEM, 10% FBS,and 1% penicillin/streptomycin) in an opaque 96-well microplate (3000cells per well) and incubated overnight. The peptide inhibitors wereadded to the cells the next day in 5 μL of assay medium and the platewas incubated at 37° c. for 2 h. After that, 5 μL of assay mediacontaining recombinant TNFα (final concentration 5 ng/mL) was added tothe wells. The plate was then returned to the incubator for 4 h at 37°c. Finally, 50 μL of ONE-Step luciferase assay reagent was added to eachwell and after 10 minutes the luminescence was measured on a TecanInfinite M1000 Pro microplate reader. Data presented were the mean±SD ofat least three independent experiments (n=6 for basal activities).

In the absence of any inhibitor, treatment of HEK 293(Luc) cells withTNFα activated the IKK complex and nuclear translocation of NF-κB,resulting in a 15-fold increase in the luciferase activity (FIG. 2C).Prior incubation of the cells with peptide 7 dose-dependently inhibitedthe TNFα-induced NF-κB activation with an IC50 value of 10 μM (FIGS. 2Cand 2D). Antp-NBD (peptide 1) also inhibited NF-κB activation, but withan IC50 of about 41 μM, in agreement with previous reports. The twonegative control peptides (peptide 20 and peptide 21) decreased theluciferase activity only at high concentrations.

It is to be noted that at very high concentrations, cationic CPPs maycause nonspecific cytotoxicity to mammalian cells and reduce theexpression of the luciferase gene. Further, at high concentrations (>10μM), CPPs can directly cross the plasma membrane through a yet poorlydefined “direct translocation” mechanism, in addition to endocyticuptake, resulting in much greater cytosolic entry efficiency. Bothfactors may cause artificially low cellular IC₅₀ values in theluciferase assay for poorly active peptides (such as Antp-NBD, peptide20 and peptide 21). As discussed earlier, NEMO is not involved in thenon-canonical NF-κB signaling pathway. A specific inhibitor against NEMOshould not block the basal NF-κB activity, which has importantphysiological functions.

To test whether peptide 7 also affects the basal NF-κB activity, theluciferase assay was repeated in the absence of TNFα stimulation. Asshown in FIG. 2C, peptide 7 did not inhibit the basal NF-κB activity.Instead, it caused a small but statistically significant increase in thebasal NF-κB activity (about 1.5-fold). Such a small stimulatory effectwas previously reported for Antp-NBD and attributed to increasedavailability of IKKα/β for the non-canonical NF-κB signaling pathwaywhen the canonical pathway is blocked. To ascertain that inhibition ofNF-κB signaling by peptide 7 is caused by disruption of the NEMO-IKKinteraction, we treated HT29 colon cancer cells with peptide 7 and TNFαand examined the levels of phosphorylated (and activated) IKKβ and IκBα,which acts immediately downstream of the NEMO-IKK complex, by westernblot analysis.

As expected, peptide 7 (O-25 μM) dose-dependently inhibited TNFα-inducedphosphorylation of IKKβ, while the total intracellular IKKβ level wasunchanged (FIG. 2E). For immunoblot analysis, HT29 cells were grown in a6-well plate to 80-90% confluency in standard DMEM (Life Technologies)supplemented with 10% FBS and 1% penicillin-streptomycin sulfate at 37°C. in 5% CO2. The cells were treated with 0, 2, 10, or 25 μM peptide 7for 2 hours followed by stimulation with TNFα (5 ng/ml) for 10 minutes.The cells were rapidly washed with cold PBS and trypsinized to detachfrom the plate. Following centrifugation and resuspension in PBS toremove any remaining trypsin the pelleted cells were lysed in 100 μl ofPierce™ RIPA Buffer (Thermo) containing protease and phosphataseinhibitors for 30 minutes on ice. Cell lysates were centrifuged at 15000rpm for 20 minutes, and the extracted proteins in the supernatant werecollected. After measuring the concentration of the samples using theBCA Protein Assay Kit (Thermo) and adjusting the total proteinconcentration to be the same for each sample, SDS-PAGE loading bufferwas added and the samples were boiled for 5 minutes. Equal amounts ofprotein were loaded onto a 10% SDS-PAGE gel (130 V, 2.5 hours) followedby electrophoretic transfer to a nitrocellulose mem-brane at 4° C. (90V, 2.5 hours). The membrane was blocked using TBST buffer (20 mM Tris pH7.5, 150 mM NaCl, 0.1% (v/v) Tween-20) containing 5% milk proteins(Bio-Rad) at room temperature for 1 h and finally the membrane wasprobed with the following rabbit monoclonal antibodies: p-IKKα(Ser176)/IKKβ (Ser177) (Cell Signaling Technologies, 2071), IKKβ (CellSignaling Technologies, 8943), IκBα (Cell Signaling Technologies, 4812),and β-Actin (Sigma, A5441). Primary antibodies were diluted according tothe suggestion of the manufacturer and incubated with the nitrocellulosemem-brane overnight at 4° C. After washing, the appropriate HRPconjugated secondary antibody was added to each membrane and incu-batedat room temperature for 2 hours. The membrane was washed with TBSTsolution again and the signals were detected with the ChemiluminescentHRP Antibody Detection Reagent (Denville, E-2500) by following themanufacturer's protocol.

Concomitantly, peptide 7 increased the intracellular level of IκBα,presumably by inhibiting TNFα-induced proteasomal degradation. The sameeffects on NF-κB signaling had previously been reported for Antp-NBD. Inagreement with the luciferase assay results (FIG. 2C), peptide 7 showedno effect on the phospho-IKKβ or IκBα level when cells were notstimulated with TNFα. Taken together, the above results indicate thatpeptide 7 efficiently enters the cytosol of mammalian cells andselectively inhibits the canonical NF-κB signaling pathway by blockingthe NEMO-IKK interaction.

In general, peptide 7 selectively inhibits the canonical NF-κB signalingpathway but not the non-canonical NF-κB pathway. Consequently, peptide 7inhibits the proliferation and survival of cancer cells with elevatedNF-κB activities but has minimal cytotoxicity to normal cells. We havedemonstrated the feasibility of designing cell-permeable bicyclicpeptides whose CPP motif performs the dual function of cell penetrationand target engagement. This strategy results in biologically activemacrocycles that are relatively small in size and have more “drug-like”properties.

Example 3. Anticancer Activity by Bicyclic Peptidyl Inhibitors

The anticancer activity of the bicyclic peptidyl inhibitors wasanalyzed. Aberrant activation of NF-κB via the canonical signalingpathway plays critical roles during the initiation and progression ofcertain cancers. For example, NF-κB is excessively activated in ovariancancer cell lines A2780 and CP70. Moreover, blocking canonical NF-κBactivation in A2780 cells resulted in apoptosis. We therefore testedpeptide 7 for potential anticancer activity against cell lines withhyperactivated NF-κB. We first examined the entry of peptide 7 intoA2780 ovarian cancer cells by live-cell confocal microscopy. Forconfocal microscopy, A2780 cells were seeded into a glass-bottomedculture dish (MatTek) and cultured overnight at 37° c. (5×10⁴ cells/mL).The next day the cells were washed with DPBS (2×) and treated with 5 μMfluorescein-labeled peptide in RPMI media containing 10% FBS, andincubated at 37° c. for 2 h. The media containing the peptide wasremoved and the cells were washed twice with phenol-free RPMI media(2×). The cells were imaged on a Visitech Infinity 3 Hawk 2D-array livecell imaging confocal microscope equipped with a 60× oil objective.

Treatment of the cells with 5 μM FITC-labeled peptide 7 for 2 h resultedin intense green fluorescence inside all cells (FIG. 4A). Next, theeffect of pep-tide 7 on the viability of A2780 and CP70 cells wasassessed by a methylene blue cell viability assay. For the methyleneblue cell viability assay, A2780 (2500 cells/well), CP70 (2000cells/well) or OSE (3000 cells/well) were seeded into a 96-wellmicroplate in 100 μL respective growth media and incubated at 37° c.overnight. The following day, the seeded cells were treated withincreasing concentrations of peptide for 72 hours. The treated cellswere washed with DPBS and fixed to the plate by treating with 3.7%formaldehyde solution for 1 hour. Fixation solution was removed and thefixed cells were treated with 1.0% methylene blue solution for 30minutes. Following staining, the plate was rinsed under running waterfor approximately five min and left to dry. Finally, 100 μL ofsolubilization buffer (10% acetic acid, 50% methanol, and 40% H2O) wasadded to each well and the absorbance was measured at 630 nm using aTecan Infinite M1000 Pro microplate reader. Data presented were themean±SD of five independent experiments.

Peptide 7 dose-dependently reduced the viability of both ovarian cancercell lines, with LD50 values of about 20 and 10 μM for A2780 and CP70cells, respectively (FIG. 4B). In contrast, peptide 7 had no significanteffect on ovarian surface epithelial cells (OSE), a non-cancerousovarian cell line with normal NF-κB activity. The greater sensitivity ofCP70 cells to peptide 7 than A2780 cells is consistent with the greaterdependency on NF-κB activity by the former. CP70 cells were derived fromA2780 cells by subjecting the latter to low, repeated doses of cisplatinand selecting for cisplatin resistant clones. Peptide 7 also reduced theviability of A375 melanoma cells, which have constitutively activatedNF-κB. The treatment of A375 cells with Antp-NBD decreased the NF-κBactivity and induced growth arrest and apoptosis was previouslydisclosed in Ianaro et al. (Cancer Lett. 2009, 274, 331-336), whichcontents is incorporated by reference in its entirety.

To determine whether the observed anticancer activity of peptide 7 iscorrelated with specific inhibition of the NEMO-IKK interaction, we alsotested the effect of peptides 1, 20, and 21 on A2780 cells by using theMTT cell viability assay. For the MTT cell viability assay, HeLa,HEK293, A2780, OSE or A375 cells were seeded into a 96-well microplate(3000 cells/well) in 100 μl of respective growth media and incubated at37° c. overnight. The next day, a serial dilution of peptide was addedto each well in 10 μL of assay medium. The treated cells were incubatedat 37° c. with 5% CO2 for 72 hours. Following compound treatment, 10 μLof MTT stock solution (Roche) was added to each well. After anadditional 4 h at 37° C., 100 μL of SDS-HCl solubilizing solution wasadded to each well and the plate was returned to the incubator overnightat 37° c. A Tecan Infinite M1000 Pro microplate reader was used thefollowing morning to measure the absorbance of the formazan product at565 nm. Data presented were the mean±SD of three independentexperiments.

Although Antp-NBD also reduced the viability of A2780 cells in adose-dependent manner, it was less active than peptide 7 (LD50 values ofabout 30 μM and >80 μM for peptide 7 and Antp-NBD, respectively; FIG.4C). This is consistent with the fact that Antp-NBD is less potent inNEMO binding, less cell-permeable, and less metabolically stable thanpeptide 7. As expected, peptides 20 and 21 were also much less activethan peptide 7 in this assay. Under the same conditions, none of thefour peptides had significant effect on the viability of non-cancerousOSE cells (FIG. 4D). These results strongly sug-gest that the observedanticancer activity of peptide 7 (and Antp-NBD) is caused by theirinhibition of the NEMO-IKK interaction and NF-κB activation.

INCORPORATION BY REFERENCE

All publications cited herein are incorporated by reference in itsentirety for all purposes.

The invention claimed is:
 1. A bicyclic polypeptide comprising Formula5A or 5B:

wherein: CPP comprises a sequence according to Formula 2:(AA_(u))_(m)-AA₁-AA₂-AA₃-AA₄-(AA_(z))_(n)  2 wherein: each of AA₁, AA₂,AA₃, AA₄, AA_(u), and AA, are independently selected from a D or L aminoacid, m and n are independently selected from a number from 0 to 6; andwherein: at least two of AA_(u), AA′, AA₂, AA₃, AA₄, and AA, areindependently arginine, and at least two of AA_(u), AA₁, AA₂, AA₃, AA₄,and AA_(z) are independently a hydrophobic amino acid; Xm is a 4-7 aminoacid peptide comprising a sequence selected from any one of GWIY (SEQ IDNO: 1), GWIYA (SEQ ID NO: 2), GWIYa (SEQ ID NO: 50), AGWIY (SEQ ID NO:3), aGWIY (SEQ ID NO: 51), AWIYA (SEQ ID NO: 4), GAIYA (SEQ ID NO: 5),GWAYA (SEQ ID NO: 6), GWIAA (SEQ ID NO: 7), GWIYA (SEQ ID NO: 8), GAIAA(SEQ ID NO: 9), GAAAA (SEQ ID NO: 10), or the inverse of any one of theaforementioned Xm sequences (SEQ ID NOs: 52-63); L is a linker moiety;and each of R₁, R₂, and R₃ independently comprise an amide, an ester, atriazole, or a combination thereof.
 2. A bicyclic polypeptide comprisingFormula 5C or 5D:

wherein: CPP comprises a sequence according to Formula 2:(AA_(u))_(m)-AA₁-AA₂-AA₃ ⁻AA₄-(AA_(z))_(n) wherein: each of AA₁, AA₂,AA₃, AA₄, AA_(u), and AA_(z) are independently selected from a D or Lamino acid, and m and n are independently selected from a number from 0to 6; and wherein: at least two of AA_(u), AA₁, AA₂, AA₃, AA₄, andAA_(z) are independently arginine, and at least two of AA_(u), AA′, AA₂,AA₃, AA₄, and AA, are independently a hydrophobic amino acid; Xm is a4-7 amino acid peptide comprising a sequence selected from any one ofGWIY (SEQ ID NO: 1), GWIYA (SEQ ID NO: 2), GWIYa (SEQ ID NO: 50), AGWIY(SEQ ID NO: 3), aGWIY (SEQ ID NO: 51), AWIYA (SEQ ID NO: 4), GAIYA (SEQID NO: 5), GWAYA (SEQ ID NO: 6), GWIAA (SEQ ID NO: 7), GWIYA (SEQ ID NO:8), GAIAA (SEQ ID NO: 9), GAAAA (SEQ ID NO: 10), or the inverse of anyone of the aforementioned Xm sequences (SEQ ID NOs: 52-63); AA_(L) ateach instance is an amino acid; p is selected from a number from 0 to 3;L is a linker moiety; and each of R₁, R₂, and R₃ comprise an amide, anester, a triazole, or a combination thereof.
 3. The bicyclic polypeptideof claim 1, wherein both the CPP sequence and the Xm sequenceparticipate in binding to NEMO.
 4. The bicyclic polypeptide of claim 1,wherein the bicyclic polypeptide binds to the IKKα/β-binding domain onNEMO.
 5. The bicyclic polypeptide of claim 1, wherein the bicyclicpolypeptide inhibits NEMO-IKKα/β interaction by at least about 10%. 6.The bicyclic polypeptide of claim 1, wherein the bicyclic polypeptidehas an IC50 of about 10 μM or less when measured for the NEMO-IKKα/βinteraction.
 7. The bicyclic polypeptide of claim 6, wherein thebicyclic polypeptide has an IC50 of about 1.0 μM or less when measuredfor the NEMO-IKKα/β interaction.
 8. The bicyclic polypeptide of claim 1,wherein the CPP comprises a sequence according to Formula 3A-D:

wherein: each of AA_(H1) and AA_(H2) are independently a hydrophobicamino acid; each of AA_(U) and AA_(Z) are independently any amino acid;and m and n are independently selected from a number from 0 to
 6. 9. Apharmaceutical composition comprising the bicyclic polypeptide of claim1 and one or more pharmaceutically acceptable excipients.
 10. A cellcomprising the bicyclic polypeptide of claim
 1. 11. The bicyclicpolypeptide of claim 2, wherein the bicyclic polypeptide has an IC50 ofabout 10 μM or less when measured for the NEMO-IKKα/β interaction.